Nucleic acid-guided editing of exogenous polynucleotides in heterologous cells

ABSTRACT

The present disclosure provides shuttle vectors for editing exogenous polynucleotides in heterologous live cells, as well as automated methods, modules, and multi-module cell editing instruments and systems for performing the editing methods.

RELATED CASES

The present invention is a continuation of U.S. Ser. No. 17/232,046,filed 15 Apr. 2021, now allowed; which is a continuation of Ser. No.16/950,878, filed 17 Nov. 2020, now U.S. Pat. No. 10,995,424; which is acontinuation of U.S. Ser. No. 16/798,338, filed 22 Feb. 2020, now U.S.Pat. No. 10,858,761 which claims priority to U.S. Ser. No. 62/810,001,filed 25 Feb. 2019; which is a continuation-in-part of U.S. Ser. No.16/670,340, filed 31 Oct. 2019, now U.S. Pat. No. 10,676,842; which is acontinuation of U.S. Ser. No. 16/392,605, filed 23 Apr. 2019, now U.S.Pat. No. 10,557,216; which claims priority to U.S. Ser. No. 62/671,266,filed 14 May 2018, and U.S. Ser. No. 62/662,126, filed 24 Apr. 2018, allof which are incorporated by reference for all purposes.

FIELD OF THE INVENTION

The present disclosure provides shuttle vectors and automated methodsand multi-module cell editing instruments and systems for editingexogenous polynucleotides in heterologous cells.

BACKGROUND OF THE INVENTION

In the following discussion certain articles and methods will bedescribed for background and introductory purposes. Nothing containedherein is to be construed as an “admission” of prior art. Applicantexpressly reserves the right to demonstrate, where appropriate, that thearticles and methods referenced herein do not constitute prior art underthe applicable statutory provisions.

In the following discussion certain articles and methods will bedescribed for background and introductory purposes. Nothing containedherein is to be construed as an “admission” of prior art. Applicantexpressly reserves the right to demonstrate, where appropriate, that thearticles and methods referenced herein do not constitute prior art underthe applicable statutory provisions.

Genome editing with engineered nucleases is a method in which changes tonucleic acids are made in the genome of a living organism. Certainnucleases create site-specific double-strand breaks at target regions inthe genome, which can be repaired by homologous recombination, resultingin targeted edits. These nucleases can be used to introduce one or moreedits into multiple cells simultaneously, allowing for the production oflibraries of cells with one or more edits in the genome. Moreover, inaddition to the genome, nucleic acid guided nuclease editing can be usedto edit exogenous nucleic acid sequences in a cell, such as nucleic acidsequences residing on a vector.

In some circumstances, it may be desirable to edit nucleic acidsequences of a cell type or genome that is difficult to manipulate,maintain in culture, or edit (e.g., a source cell or genome). In suchcircumstances a shuttle vector may be useful for cloning the targetpolynucleotide and editing the sequence in a cell type that is easy tomanipulate, maintain in culture and edit due to, e.g., the cell typebeing well-studied and well-known in the art (e.g. an editing cell).Once edited, the shuttle vector with the edited target polynucleotidemay be returned to the source cell type for further study.

There are various and diverse methods for delivering exogenouspolynucleotides from the genome of one organism to another, in this casefor automated editing by nucleic acid-guided nucleases, and a number ofconsiderations must be taken into account when designing the methods andshuttle vectors to be used, including payload size of the exogenouspolynucleotide locus; means for maintaining the shuttle vector at asuitable copy number during expansion and propagation of the sourcecells and editing cells; specific requirements of transformation,transfection, and nucleic acid isolation protocols depending on thespecies of the source cells and editing cells; and the ultimatedestination of the edited target polynucleotide.

To date, however, editing of nucleic acids—much less shuttle vectors orlarger artificial chromosomes carrying hundreds of kilobases ofexogenous polynucleotides—in a cell has not been compatible withautomation due to low efficiencies and challenges with cell selection.There is thus a need for shuttle vectors and automated methods andmulti-module cell processing instruments capable of editing exogenouspolynucleotides in heterologous cells. The present invention addressesthis need.

SUMMARY OF THE INVENTION

This Summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This Summary is not intended to identify key or essentialfeatures of the claimed subject matter, nor is it intended to be used tolimit the scope of the claimed subject matter. Other features, details,utilities, and advantages of the claimed subject matter will be apparentfrom the following written Detailed Description including those aspectsillustrated in the accompanying drawings and defined in the appendedclaims.

The present disclosure provides shuttle vectors carrying inserts oftarget polynucleotides and automated modules and multi-module cellediting instrumentation for nucleic-acid guided nuclease editing oftarget polynucleotides in heterologous editing cells. The shuttlevectors are used to transfer target polynucleotides from cells of onesource organism to cells of a heterologous editing organism. Nucleicacids comprising editing “machinery” are introduced into the editingcells to enable nucleic acid-guided nuclease editing of the targetpolynucleotides. Edited target polynucleotides are subsequentlycharacterized, isolated, and used for downstream applications, includingre-introduction of the edited polynucleotides into cells of the sourceorganism.

Nucleic-acid guided nuclease editing of target polynucleotides is apowerful tool for research, biochemical production, cell engineering andtherapy, where automated methods and modules and multi-module cellediting instrumentation for nucleic-acid guided nuclease editing presentdistinct advantages over the state of the art. The present disclosureenables automated nucleic-acid guided nuclease editing of targetpolynucleotides from the genome of essentially any organism.

Thus, there is provided a method of automated nucleic acid-guidednuclease editing of exogenous polynucleotides from source cells withinheterologous editing cells, comprising: inserting one or more targetpolynucleotides from the source cells into shuttle vector backbones toform a library of shuttle vectors; transferring the library of shuttlevectors into a first receptacle; providing heterologous editing cells ina second receptacle; growing the heterologous editing cells in a growthmodule; transferring the heterologous editing cells from the growthmodule to a cell concentration module; concentrating and renderingelectrocompetent the heterologous editing cells in the cellconcentration module; introducing the library of shuttle vectors intothe heterologous editing cells in a transformation module; providing oneor more editing vectors wherein the editing vectors comprise a codingsequence for a nuclease, a guide nucleic acid and a DNA donor sequencein a third receptacle; introducing the one or more editing vectors intothe heterologous editing cells in the transformation module;transferring the heterologous editing cells from the transformationmodule to an editing module; allowing editing to take place in theediting module under conditions that allow the editing vectors to editthe one or more target polynucleotides in the shuttle vectors therebyforming edited shuttle vectors; identifying living editing cellscontaining the edited shuttle vectors; isolating the living editingcells containing the edited shuttle vectors; isolating the editedshuttle vectors; wherein all of the first receptacle, second receptacle,third receptacle, growth module, cell concentration module,transformation module and editing module are comprised within anautomated multi-module cell processing instrument. In some aspects, aliquid handling system moves the heterologous editing cells from thefirst receptacle to the growth module, from the growth module to thecell concentration module, from the cell concentration module to thetransformation module, and from the transformation module to the editingmodule; and moves the shuttle vector library from the second receptacleto the transformation module and moves the one or more editing vectorsfrom the third receptacle to the transformation module.

In some aspects, the heterologous editing cells are bacterial cells; insome aspects, the heterologous editing cells are yeast cells; and insome aspects the heterologous editing cells are mammalian cells. In someaspects, the heterologous editing cells are iPSCs.

In some aspects, the shuttle vector backbone is a bacterial plasmid thatcomprises a DNA plasmid comprising a bacterial origin of replication andselectable marker, and in some aspects, the shuttle vector is abacterial artificial chromosome.

In other aspects, the shuttle vector backbone is a yeast plasmid thatcomprises a DNA plasmid comprising a yeast origin of replication, anARS, CEN sequence, and selectable marker, and in some aspects, theshuttle vector is a yeast artificial chromosome. In yet other aspects,the shuttle vector backbone is a synthetic chromosome.

In some aspects, at least one target polynucleotide is selected from afull-length gene; an open reading frame; or a genomic locus of size1000-10,000 nucleotides, 50-500 nucleotides, 10-100 nucleotides, or10,000-100,000 nucleotides.

In some aspects, the source cells are animal cells and in some aspects,the animal cells are mammalian cells. In some aspects, the mammaliancells are human cells. In other aspects, the source cells are bacterialcells. In yet other aspects, the source cells are yeast cells or plantcells.

In some aspects, the shuttle vector transfers nucleic acids from onespecies of bacteria to another species of bacteria, or from one speciesof yeast to another species of yeast, or from one species of eukaryoteto another species of eukaryote, or from bacteria to yeast then toanimal cells, or from yeast to plants, or from plants to yeast, or fromyeast to animal cells, or from animal cells to yeast, or from one typeof animal cell to another type of animal cell from the same animal, orfrom one species of animal to another species of animal.

These aspects and other features and advantages of the invention aredescribed below in more detail.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features and advantages of the present inventionwill be more fully understood from the following detailed description ofillustrative embodiments taken in conjunction with the accompanyingdrawings in which:

FIG. 1 is a process diagram showing the various pathways for buildingand editing shuttle vectors.

FIGS. 2A-2C depict three different views of an exemplary automatedmulti-module cell processing instrument for performing nucleicacid-guided nuclease editing.

FIG. 3A depicts one embodiment of a rotating growth vial for use withthe cell growth module described herein and in relation to FIGS. 3B-3D.FIG. 3B illustrates a perspective view of one embodiment of a rotatinggrowth vial in a cell growth module housing. FIG. 3C depicts a cut-awayview of the cell growth module from FIG. 3B. FIG. 3D illustrates thecell growth module of FIG. 3B coupled to LED, detector, and temperatureregulating components.

FIG. 4A depicts retentate (top) and permeate (bottom) members for use ina tangential flow filtration module (e.g., cell growth and/orconcentration module), as well as the retentate and permeate membersassembled into a tangential flow assembly (bottom).

FIG. 4B depicts two side perspective views of a reservoir assembly of atangential flow filtration module. FIGS. 4C-4E depict an exemplary top,with fluidic and pneumatic ports and gasket suitable for the reservoirassemblies shown in FIG. 4B.

FIG. 5A depicts an exemplary combination reagent cartridge andelectroporation device (e.g., transformation module) that may be used ina multi-module cell processing instrument. FIG. 5B is a top perspectiveview of one embodiment of an exemplary flow-through electroporationdevice that may be part of a reagent cartridge.

FIG. 5C depicts a bottom perspective view of one embodiment of anexemplary flow-through electroporation device that may be part of areagent cartridge. FIGS. 5D-5F depict a top perspective view, a top viewof a cross section, and a side perspective view of a cross section of anFTEP device useful in a multi-module automated cell processinginstrument such as that shown in FIGS. 2A-2C.

FIG. 6A depicts a simplified graphic of a workflow for singulating,editing and normalizing cells in a solid wall device. FIGS. 6B-6D depictan embodiment of a solid wall isolation incubation and normalization(SWIIN) module. FIG. 6E depicts the embodiment of the SWIIN module inFIGS. 6B-6D further comprising a heater and a heated cover.

FIG. 7 is a simplified block diagram of an embodiment of an exemplaryautomated multi-module cell processing instrument comprising a solidwall singulation/growth/editing/normalization module, in this case, usedfor recursive editing.

FIG. 8 is an example control system for use in an automated multi-modulecell editing instrument.

FIG. 9 is a table showing the edit category and the number of wells thatwere in each edit category obtained for the experiment described inExample 4.

FIG. 10 is a bar graph showing edit categories obtained for theexperiment described in Example 4.

It should be understood that the drawings are not necessarily to scale,and that like reference numbers refer to like features.

DETAILED DESCRIPTION

All of the functionalities described in connection with one embodimentof the methods, devices or instruments described herein are intended tobe applicable to the additional embodiments of the methods, devices andinstruments described herein except where expressly stated or where thefeature or function is incompatible with the additional embodiments. Forexample, where a given feature or function is expressly described inconnection with one embodiment but not expressly mentioned in connectionwith an alternative embodiment, it should be understood that the featureor function may be deployed, utilized, or implemented in connection withthe alternative embodiment unless the feature or function isincompatible with the alternative embodiment.

The practice of the techniques described herein may employ, unlessotherwise indicated, conventional techniques and descriptions ofmolecular biology (including recombinant techniques), cell biology,biochemistry, and genetic engineering technology, which are within theskill of those who practice in the art. Such conventional techniques anddescriptions can be found in standard laboratory manuals such as Greenand Sambrook, Molecular Cloning: A Laboratory Manual. 4th, ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (2014);Current Protocols in Molecular Biology, Ausubel, et al. eds., (2017);Neumann, et al., Electroporation and Electrofusion in Cell Biology,Plenum Press, New York, 1989; and Chang, et al., Guide toElectroporation and Electrofusion, Academic Press, California (1992),all of which are herein incorporated in their entirety by reference forall purposes. For techniques for creating synthetic chromosomes and celltransformation and growth, see Cell and Tissue Culture: LaboratoryProcedures in Biotechnology (Doyle & Griffiths, eds., John Wiley & Sons1998); Mammalian Chromosome Engineering—Methods and Protocols (G.Hadlaczky, ed., Humana Press 2011), both of which are hereinincorporated by reference in their entirety for all purposes. Nucleicacid-guided nuclease-specific techniques can be found in, e.g., GenomeEditing and Engineering From TALENs and CRISPRs to Molecular Surgery,Appasani and Church, 2018; and CRISPR: Methods and Protocols, Lindgrenand Charpentier, 2015; both of which are herein incorporated in theirentirety by reference for all purposes.

Note that as used herein and in the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a cell” refers toone or more cells, and reference to “the system” includes reference toequivalent steps, methods and devices known to those skilled in the art,and so forth. Additionally, it is to be understood that terms such as“left,” “right,” “top,” “bottom,” “front,” “rear,” “side,” “height,”“length,” “width,” “upper,” “lower,” “interior,” “exterior,” “inner,”“outer” that may be used herein merely describe points of reference anddo not necessarily limit embodiments of the present disclosure to anyparticular orientation or configuration. Furthermore, terms such as“first,” “second,” “third,” etc., merely identify one of a number ofportions, components, steps, operations, functions, and/or points ofreference as disclosed herein, and likewise do not necessarily limitembodiments of the present disclosure to any particular configuration ororientation.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All publications mentionedherein are incorporated by reference for the purpose of describing anddisclosing devices, formulations and methodologies that may be used inconnection with the presently described invention.

Where a range of values is provided, it is understood that eachintervening value, between the upper and lower limit of that range andany other stated or intervening value in that stated range isencompassed within the invention. The upper and lower limits of thesesmaller ranges may independently be included in smaller ranges, and arealso encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

In the following description, numerous specific details are set forth toprovide a more thorough understanding of the present invention. However,it will be apparent to one of skill in the art that the presentinvention may be practiced without one or more of these specificdetails. In other instances, features and procedures well known to thoseskilled in the art have not been described in order to avoid obscuringthe invention. The terms used herein are intended to have the plain andordinary meaning as understood by those of ordinary skill in the art.

A “centromere” is any nucleic acid sequence that confers an ability of achromosome to segregate to daughter cells through cell division. Acentromere may confer stable segregation of a nucleic acid sequence,including a synthetic chromosome containing the centromere, throughmitotic and meiotic divisions. A centromere does not necessarily need tobe derived from the same species as the cells into which it isintroduced, but preferably the centromere has the ability to promote DNAsegregation in cells of that species. A “dicentric” chromosome is achromosome that contains two centromeres. A “formerly dicentricchromosome” is a chromosome that is produced when a dicentric chromosomefragments. A “chromosome” is a nucleic acid molecule—and associatedproteins—that is capable of replication and segregation in a cell upondivision of the cell. Typically, a chromosome contains a centromericregion, replication origins, telomeric regions and a region of nucleicacid between the centromeric and telomeric regions. An “acrocentricchromosome” refers to a chromosome with arms of unequal length.

A “coding sequence” or a sequence that “encodes” a peptide is a nucleicacid molecule that is transcribed (in the case of DNA) and translated(in the case of mRNA) into a polypeptide in vivo when placed under thecontrol of appropriate control sequences. The boundaries of the codingsequence typically are determined by a start codon at the 5′ (amino)terminus and a translation stop codon at the 3′ (carboxy) terminus.

The term “complementary” as used herein refers to Watson-Crick basepairing between nucleotides and specifically refers to nucleotideshydrogen-bonded to one another with thymine or uracil residues linked toadenine residues by two hydrogen bonds and cytosine and guanine residueslinked by three hydrogen bonds. In general, a nucleic acid includes anucleotide sequence described as having a “percent complementarity” or“percent homology” to a specified second nucleotide sequence. Forexample, a nucleotide sequence may have 80%, 90%, or 100%complementarity to a specified second nucleotide sequence, indicatingthat 8 of 10, 9 of 10 or 10 of 10 nucleotides of a sequence arecomplementary to the specified second nucleotide sequence. For instance,the nucleotide sequence 3′-TCGA-5′ is 100% complementary to thenucleotide sequence 5′-AGCT-3′; and the nucleotide sequence 3′-TCGA-5′is 100% complementary to a region of the nucleotide sequence5′-TAGCTG-3′.

The term DNA “control sequences” refers collectively to promotersequences, polyadenylation signals, transcription termination sequences,upstream regulatory domains, origins of replication, internal ribosomeentry sites, nuclear localization sequences, enhancers, and the like,which collectively provide for the replication, transcription andtranslation of a coding sequence in a recipient cell. Not all of thesetypes of control sequences need to be present so long as a selectedcoding sequence is capable of being replicated, transcribed and—for somecomponents—translated in an appropriate host cell.

As used herein the term “donor DNA” or “donor nucleic acid” refers tonucleic acid that is designed to introduce a DNA sequence modification(insertion, deletion, substitution) into a locus (e.g., a target genomicDNA sequence or cellular target sequence) by homologous recombinationusing nucleic acid-guided nucleases. For homology-directed repair, thedonor DNA must have sufficient homology to the regions flanking the “cutsite” or site to be edited in the genomic target sequence. The length ofthe homology arm(s) will depend on, e.g., the type and size of themodification being made. In many instances and preferably, the donor DNAwill have two regions of sequence homology (e.g., two homology arms) tothe genomic target locus. Preferably, an “insert” region or “DNAsequence modification” region—the nucleic acid modification that onedesires to be introduced into a genome target locus in a cell-will belocated between two regions of homology. The DNA sequence modificationmay change one or more bases of the target genomic DNA sequence at onespecific site or multiple specific sites. A change may include changing1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300,400, or 500 or more base pairs of the genomic target sequence. Adeletion or insertion may be a deletion or insertion of 1, 2, 3, 4, 5,10, 15, 20, 25, 30, 40, 50, 75, 100, 150, 200, 300, 400, or 500 or morebase pairs of the genomic target sequence.

“Endogenous chromosomes” refer to chromosomes found in a cell prior togeneration or introduction of exogenous genetic material.

The terms “guide nucleic acid” or “guide RNA” or “gRNA” refer to apolynucleotide comprising 1) a guide sequence capable of hybridizing toa genomic target locus, and 2) a scaffold sequence capable ofinteracting or complexing with a nucleic acid-guided nuclease.

“Homology” or “identity” or “similarity” refers to sequence similaritybetween two peptides or, more often in the context of the presentdisclosure, between two nucleic acid molecules. The term “homologousregion” or “homology arm” refers to a region on the donor DNA with acertain degree of homology with the target genomic DNA sequence.Homology can be determined by comparing a position in each sequencewhich may be aligned for purposes of comparison. When a position in thecompared sequence is occupied by the same base or amino acid, then themolecules are homologous at that position. A degree of homology betweensequences is a function of the number of matching or homologouspositions shared by the sequences.

“Operably linked” refers to an arrangement of elements where thecomponents so described are configured so as to perform their usualfunction. Thus, control sequences operably linked to a coding sequenceare capable of effecting the transcription, and in some cases, thetranslation, of a coding sequence. The control sequences need not becontiguous with the coding sequence so long as they function to directthe expression of the coding sequence. Thus, for example, interveninguntranslated yet transcribed sequences can be present between a promotersequence and the coding sequence and the promoter sequence can still beconsidered “operably linked” to the coding sequence. In fact, suchsequences need not reside on the same contiguous DNA molecule (i.e.chromosome) and may still have interactions resulting in alteredregulation.

As used herein, the terms “protein” and “polypeptide” are usedinterchangeably. Proteins may or may not be made up entirely of aminoacids.

A “promoter” or “promoter sequence” is a DNA regulatory region capableof binding RNA polymerase and initiating transcription of apolynucleotide or polypeptide coding sequence such as messenger RNA,ribosomal RNA, small nuclear or nucleolar RNA, guide RNA, or any kind ofRNA transcribed by any class of any RNA polymerase I, II or III.Promoters may be constitutive or inducible.

As used herein the term “selectable marker” refers to a gene introducedinto a cell, which confers a trait suitable for artificial selection.General use selectable markers are well-known to those of ordinary skillin the art. Drug selectable markers such as ampicillin/carbenicillin,kanamycin, chloramphenicol, erythromycin, tetracycline, gentamicin,bleomycin, streptomycin, puromycin, hygromycin, blasticidin, and G418may be employed. In other embodiments, selectable markers include, butare not limited to human nerve growth factor receptor (detected with aMAb, such as described in U.S. Pat. No. 6,365,373); truncated humangrowth factor receptor (detected with MAb); mutant human dihydrofolatereductase (DHFR; fluorescent MTX substrate available); secreted alkalinephosphatase (SEAP; fluorescent substrate available); human thymidylatesynthase (TS; confers resistance to anti-cancer agentfluorodeoxyuridine); human glutathione 5-transferase alpha (GSTA1;conjugates glutathione to the stem cell selective alkylator busulfan;chemoprotective selectable marker in CD34+ cells); CD24 cell surfaceantigen in hematopoietic stem cells; human CAD gene to confer resistanceto N-phosphonacetyl-L-aspartate (PALA); human multi-drug resistance-1(MDR-1; P-glycoprotein surface protein selectable by increased drugresistance or enriched by FACS); human CD25 (IL-2a; detectable byMab-FITC); Methylguanine-DNA methyltransferase (MGMT; selectable bycarmustine); rhamnose, and Cytidine deaminase (CD; selectable by Ara-C).“Selective medium” as used herein refers to cell growth medium to whichhas been added a chemical compound or biological moiety that selects foror against selectable markers.

The term “specifically binds” as used herein includes an interactionbetween two molecules, e.g., an engineered peptide antigen and a bindingtarget, with a binding affinity represented by a dissociation constantof about 10⁻⁷ M, about 10⁻⁸ M, about 10⁻⁹ M, about 10⁻¹⁰ M, about 10⁻¹¹M, about 10⁻¹² M, about 10⁻¹³ M, about 10⁻¹⁴ M or about 10⁻¹⁵ M.

“Synthetic chromosomes” (also referred to in the art as “artificialchromosomes”) are nucleic acid molecules, typically DNA, that stablyreplicate and segregate alongside endogenous chromosomes in cells thathave the capacity to accommodate and express heterologous genes. A“bacterial artificial chromosome (BAC)” is a DNA construct capable ofextrachromosomal replication and segregation in bacterial cells. A“yeast artificial chromosome (YAC)” is a DNA construct capable ofextrachromosomal replication and segregation in yeast cells. A“mammalian synthetic chromosome (MAC)” refers to chromosomes that havean active mammalian centromere(s). A “human synthetic chromosome (HAC)”refers to a chromosome that includes a centromere that functions inhuman cells and that preferably is produced in human cells. Forexemplary artificial chromosomes, see, e.g., U.S. Pat. Nos. 8,389,802;7,521,240; 6,025,155; 6,077,697; 5,891,691; 5,869,294; 5,721,118;5,712,134; 5,695,967; and 5,288,625 and published International PCTapplication Nos, WO 97/40183 and WO 98/08964, all of which are hereinincorporated by reference.

The terms “target genomic DNA sequence”, “cellular target sequence”,“target sequence”, “target cellular locus” or “genomic target locus”refer to any locus in vitro or in vivo, or in a nucleic acid (e.g.,genome or episome) of a cell or population of cells, in which a changeof at least one nucleotide is desired using a nucleic acid-guidednuclease editing system. The target sequence can be a genomic locus orextrachromosomal locus. The term “edited target sequence” or “editedlocus” refers to a target genomic sequence or target sequence afterediting has been performed, where the edited target sequence comprisesthe desired edit.

The term “variant” may refer to a polypeptide or polynucleotide thatdiffers from a reference polypeptide or polynucleotide but retainsessential properties. A typical variant of a polypeptide differs inamino acid sequence from another reference polypeptide. Generally,differences are limited so that the sequences of the referencepolypeptide and the variant are closely similar overall and, in manyregions, identical. A variant and reference polypeptide may differ inamino acid sequence by one or more modifications (e.g., substitutions,additions, and/or deletions). A variant of a polypeptide may be aconservatively modified variant. A substituted or inserted amino acidresidue may or may not be one encoded by the genetic code (e.g., anon-natural amino acid). A variant of a polypeptide may be naturallyoccurring, such as an allelic variant, or it may be a variant that isnot known to occur naturally.

A “vector” is any of a variety of nucleic acids that comprise a desiredsequence or sequences to be delivered to and/or expressed in a cell.Vectors are typically composed of DNA, although RNA vectors are alsoavailable. Vectors include, but are not limited to, plasmids, fosmids,phagemids, virus genomes, synthetic chromosomes, BACs, YACs, PACs, MACs,HACs, and the like. As used herein, the term “shuttle vector” refers toa vector that is meant to be used to transfer nucleic acids of interestbetween at least two different types of living cells, such as but notlimited to a transfer of nucleic acids from one species of bacteria toanother species of bacteria, from one species of yeast to anotherspecies of yeast, from one species of eukaryote to another species ofeukaryote, from bacteria to yeast then to animal cells, from yeast toplants, from plants to yeast, from yeast to animal cells, from one typeof animal cell to another type of animal cell from the same animal, fromone species of animal to another species of animal, and any permutationor combination of these types of cells. In some embodiments of thepresent methods, two vectors—an engine vector, comprising the codingsequences for a nuclease, and an editing vector, comprising the gRNAsequence and the donor DNA sequence—are used. In alternativeembodiments, all editing components, including the nuclease, gRNAsequence, and donor DNA sequence are all on the same vector (e.g., acombined editing/engine vector).

For the editing components, as used herein, the phrase “engine vector”comprises a coding sequence for a nuclease to be used in the nucleicacid-guided nuclease systems and methods of the present disclosure. Theengine vector may also comprise, in a bacterial system, the λ Redrecombineering system or an equivalent thereto, as well as a selectablemarker. As used herein the phrase “editing vector” comprises a donornucleic acid, including an alteration to the target sequence whichprevents nuclease binding at a PAM or spacer in the target sequenceafter editing has taken place, and a coding sequence for a gRNA underthe control of a promoter. The editing vector may also comprise aselectable marker and/or a barcode. In some embodiments, the enginevector and editing vector may be combined; that is, the contents of theengine vector may be found on the editing vector such that allcomponents of the exogenous “editing machinery” are on a single vector.

Nucleic Acid-Guided Nuclease Editing in Shuttle Vectors Source GenomicLoci

The source polynucleotides for automated nucleic acid-guided nucleaseediting within a heterologous editing cell may be of varying size. Forinterrogating the function of a single gene, it may be desirable to editonly the protein-coding sequence of said gene. To enable editing of theprotein-coding sequence of a gene, in some implementations, the openreading frame (ORF) beginning with the initiation codon, ending with thetermination codon, and lacking introns, is introduced on a shuttlevector to heterologous editing cells for automated nucleic acid-guidednuclease editing. This coding sequence is typically preceded by one ofany number of desired promoters and followed by one of any number ofterminator sequences. Varieties of edits to the coding sequence of asingle gene may include saturation mutagenesis, premature terminationcodons, missense mutations, nonsense mutations, synonymous mutations,loss of function mutations, gain of function mutations, or insertions ordeletions (indels) of size ranging from 1-10 nucleotides (nt), 5-50 nt,25-100 nt, 50-500 nt, 100-1000 nt, 500-5000 nt, 1000-10,000 nt,5000-50,000 nt, or greater. In addition, source polynucleotides may be achemically-synthesized sequence derived from or inspired by (e.g.,codon-optimized, gene-fusions, operon refactoring, sequence constructsof mixed heterologous organism sources, or cDNAs) a naturally-occurringsequence and delivered into the shuttle vector or editing host as“synthetic” DNA.

To interrogate the function of an entire gene including its introns anduntranslated regions, in some implementations, the entire nucleotidesequence of a gene, coding and noncoding, is included in the shuttlevector for automated nucleic acid-guided nuclease editing withinheterologous editing cells. Exons, introns, and the borders betweenexons and introns contain information to direct accurate splicing aftertranscription. These include exonic splicing enhancer sequences,intronic splicing enhancer sequences, and alternative splicing donor andacceptor sequences. Furthermore, noncoding regions of genes, includingintrons, may encode functional noncoding RNAs, which may be a desirabletarget polynucleotide for editing. These may include microRNAs, shortinterfering RNAs, piwi-interacting RNAs, or long noncoding RNAs. The 5′untranslated regions (UTRs) of genes include essential regulatoryelements that direct processes related to transcription, translation,mRNA export, and mRNA structure. The 3′ UTRs of genes have essentialroles in regulation of transcription, translation, mRNA stability,polyadenylation, translation efficiency, post-transcriptional regulationof gene expression, and sometimes contain microRNA response elements.For all these reasons, noncoding regions of genes including the 5′ UTR,introns, and 3′ UTRs may be desirable target polynucleotides forautomated nucleic acid-guided nuclease editing within heterologousediting cells.

Genes are often subjected to long-range regulation exceeding theboundaries of their 5′ and 3′ UTRs. Long-range acting regulatoryelements may include enhancers, repressors, insulators, or mediators oflong-range epigenetic activation or silencing. For the purposes ofinterrogating the function of a gene including its long-range regulatoryelements, in some implementations, a relatively large segment ofexogenous genetic material is subjected to automated nucleic acid-guidednuclease editing within living cells. Thus, capturing a locus exceedingthe boundaries of a gene, of size 100-1000 nt, 500-5000 nt, 1000-10,000nt, 5000-50,000 nt, 10,000-100,000 nt, 50,000-500,000 nt, 100-1,000,000nt, 500,000-5,000,000 nt in a shuttle vector for automated nucleicacid-guided nuclease editing within heterologous cells may be desirable.Such a large locus will include coding regions, noncoding regions, andregulatory elements of one or many genes and may be subjected to any ofthe same sorts of mutagenesis experiments as described above.

Alternatively, in some implementations, an entire exogenous genome maybe subjected to automated nucleic acid-guided nuclease editing withinheterologous editing cells. An entire genome may be introduced to ashuttle vector in the form of a library. The choice of shuttle vector tobe used may be determined based on the destination and application(discussed infra). Methods of introduction of the genomic library to theshuttle vector may be determined based on one or more of source cellsand genome, destination, and application (discussed infra). Automatednucleic acid-guided nuclease editing of an entire exogenous genomeallows for systematic genome-wide screens of gene function, gene-by-geneinteractions, suppressor screens, user-directed evolution, genome-widescreens for genes affecting a particular phenotype, forward geneticscreens, among many other experimental approaches.

Source Genomes

Exogenous polynucleotides for automated nucleic acid-guided nucleaseediting within heterologous editing cells, in the various embodimentsdescribed herein, may be derived from the genomes of any number ofsource prokaryotic or eukaryotic organisms, including bacteria, fungi,protists, worms, insects, amphibians, fish, or mammals. The followingparagraphs discuss examples of source genomes as well as some methodsand considerations in preparing material from such genomes forintroduction into an automated nucleic acid-guided nuclease editingsystem.

Many bacterial species are useful for commercial or biomedicalapplications, or as subjects of basic, translational, or clinicalscientific inquiry. Thus, genes and genomic loci (described above) foundin bacterial genomes and plasmids are attractive targets for automatednucleic acid-guided nuclease editing within living cells. As describedabove, target loci may range in size from single genes, to largemulti-gene loci, to entire bacterial genomes. Commercial applications ofbacterial genes and pathways include production of surfactants (Desai &Banat, Fuel Energy Abstr, 38(4):221 (1997)), production of biofuels(Peralta-Yahya, et al., Nature, 488(7411):320-328 (2012)), novelproduction methods for commodity and specialty compounds (Steen, et al.,Nature, 463(7280):559-562 (2010)), biosynthesis ofpharmaceutically-useful compounds (Yuzawa, et al., Biochemistry,51(49):9779-9781 (2012)), or as bioremediation agents (U.S. Pat. No.8,440,423).

Source bacterial species from which genetic material may be derived forautomated nucleic acid-guided nuclease editing include, but are notlimited to, Escherichia coli, Shewanella oneidensis, Bacillus subtilis,Micrococcus luteus, Streptomyces aizunensis, Mycobacterium tuberculosis,Streptomyces coelicolor, Mycobacterium smegmatis, Pseudomonas putida,Desulfovibrio vulgaris, Lactobacillus acidophilus, Corynebacteriumglutamicum, Bacillus thuringiensis, Acetobacter aceti, Klebsiellapneumoniae, and Methylococcus capsulatus.

Most bacterial genomes include a single closed loop of nucleic acidsequence between approximately 500,000 nucleotides and 10,000,000nucleotides in length. Genomic DNA may be prepared by standardtechniques and target loci may be amplified or isolated and introducedto shuttle vectors according to techniques discussed supra.Alternatively, target loci may reside on bacterial plasmid DNA. PlasmidDNA may be prepared by standard techniques and target loci are amplifiedor isolated and introduced to shuttle vectors according to techniquesdiscussed supra. For both genomic targets and plasmid targets, thechoice of shuttle vector depends on target size, destination, andapplication.

Like bacteria, fungal organisms are useful for industrial applications,biomedical and biopharmaceutical applications, and as tools for basic,translational, and clinical research. Fungal genes and genomes have beenutilized in the discovery and biosynthesis of natural products (Luo, etal., Curr Opin Biotechnol., 30:230-237 (2014)), biofuels (Runguphan &Keasling, Metab Eng., 21:103-113 (2014)), pharmaceutical compounds(Punt, et al., TRENDS Biotechnol., 20(5):200-206 (2002)), and as agentsin bioremediation (U.S. Pat. No. 6,150,157). As described above, targetloci may range in size from single genes, to large multi-gene loci, toentire fungal genomes. Genes, multi-gene loci, and genomes of manyfungal organisms present attractive target polynucleotides for automatednucleic acid-guided nuclease editing in heterologous cells.

Source fungal species from which genetic material may be derived forautomated nucleic acid-guided nuclease editing include, but are notlimited to, Saccharomyces cerevisiae, Schizosaccharomyces pombe,Aspergillus oryzae, Aspergillus terreus, Aspergillus niger, Aspergillusnidulans, Trichoderma reesei, Fusarium venenatum, Cephalosporium sp.,Penicillium sp., Neurospora crassa, Yarrowia lipolytica, Pichiapastoris, and Phanerochaete chrysosporium.

Protists are a diverse group of unicellular eukaryotic microorganismsincluding flagellates, ciliates, amoebae, sporozoae, some algae, andslime molds. One genus of slime molds in particular, Dictyostelium hasbeen useful as a model laboratory organism, of which the species D.discoideum is the best-studied. Dictyostelium are commonly used to studybiological processes including cell differentiation, chemotaxis,apoptosis, thermotaxis, and as a model for Legionella infection.Dictyostelium represents a laboratory model that is intermediate incomplexity between eukaryotic unicellular fungi such as S. cerevisiae,and more complex multicellular eukaryotic genomes. The Dictyosteliumgenome contains orthologs of human disease genes which are useful forstudying pathophysiological processes including cellular differentiationand cancer metastasis and invasion. The Dictyostelium genome alsocontains an abundance of genes encoding polyketide synthases (Eichinger,et al., Nature, 435(7038):43-57 (2005)), proteins essential to thebiosynthesis and export of many natural products including someantibiotics and anticancer agents. As a useful model organism,Dictyostelium genes, multi-gene loci, and genomes are attractive targetpolynucleotides for automated nucleic acid-guided nuclease editing inheterologous cells. Genomic DNA may be isolated according to standardtechniques (Pilcher, et al., Nat Protoc, 2(6):1325-1328 (2007)) andtarget loci may be amplified or isolated and introduced to shuttlevectors according to techniques described below. Choice of shuttlevector depends on target size, destination, and application.

Increasingly complex multicellular eukaryotes introduce distinctadvantages as model organisms in the laboratory and the genes,multi-gene loci, and genomes of these organisms are thus targetpolynucleotides for automated nucleic acid-guided nuclease editing inheterologous cells. The roundworm Caenorhabditis elegans is a simplemulticellular eukaryote that has been studied extensively as a modelorganism. The 959 cells of the adult worm have been comprehensivelylineage-mapped and its simple nervous system has been a model forneuronal differentiation and innervation. The worms' advantages as amodel organism include are low cost and ease of culturing andpropagation, they are transparent, can be frozen and remain viable uponthawing, and are nonpathogenic. The genes, multi-gene loci, and genomesof C. elegans and other simple multicellular eukaryotes are possibletarget polynucleotides for automated nucleic acid-guided nucleaseediting in heterologous cells.

The genome of C. elegans is 97 Mb in size and is predicted to containapproximately 19,000 genes, many of which find homologs in more complexeukaryotes including humans (The C. elegans Sequencing Consortium,Science, 282(5396):2012-2018 (1998)). Genomic DNA may be isolatedaccording to standard techniques and target loci are amplified orisolated and introduced to shuttle vectors according to techniquesdescribed below. The choice of shuttle vector depends on target size,destination, and application.

Several insect species have been useful as model laboratory organismsand insects as a subphylum include more than one million describedspecies and include more than half of all known living organisms.Insects have significant roles and uses in agriculture, industry,ecology, and as vectors for human disease, and thus insect genes,multi-gene loci, and genomes are possible target polynucleotides forautomated nucleic acid-guided nuclease editing in heterologous cells.

In the laboratory, the fly species Drosophila melanogaster has played acritical role in the study of genetics, physiology, evolution, cellulardifferentiation and development, and microbial pathogenesis.Drosophila's advantages as a model organism include short generationtime, low cost propagation and colony maintenance, and prolific layingof macroscopically visible and manipulatable eggs. Laboratory derivedmutant strains have easily traceable phenotypes that allow for robuststudy of Mendelian genetics.

The genome of Drosophila melanogaster is approximately 140 Mb in sizeand contains four pairs of chromosomes and approximately 14,000protein-coding genes (Halligan and Keightley, Genome Res.16(2001):875-884 (2006)). Genomic DNA may be isolated according tostandard techniques and target polynucleotides may be amplified orisolated and introduced to shuttle vectors according to techniquesdescribed below. The choice of shuttle vector depends on target size,destination, and application.

In addition to Drosophila melanogaster the genes, multi-gene loci, andgenomes of many other insects may be attractive target polynucleotidesfor automated nucleic acid-guided nuclease editing in heterologouscells. Many mosquito species are vectors for human disease and may betargets for editing, including, but not limited to genera Aedes,Anopheles, Culex, Culiseta, Mansonia, Coquillettidia, Psorophora, andToxorhynchites. Agriculturally important insects include, but are notlimited to, the Brown Marmorated Stinkbug (Halyomorpha halys), AgricanFig Fly (Zaprionus indianus), Western Bean Cut Worm (Richia albicosta),Spotted Wing Drosophila (Drosophila suzukii), Elm Bark Beetles (generaHylurgopinus, Scolytus, and Pteleobius), and other insects that areeither advantages or disadvantages to agriculture.

Several mammalian species have been indispensable research tools asmodel organisms for studying human disease. As higher order eukaryoteswith increasingly complex genomes, mammalian species share homologousgenes and are far better tools for interrogating the immune, endocrine,nervous, cardiovascular, skeletal and other complex physiologicalsystems that mammals share, than are simpler model organisms. Mice,dogs, and other species naturally develop disease phenotypes affectinghumans, including cancer, atherosclerosis, hypertension, diabetes,osteoporosis, glaucoma, among others. Model organisms can also bemanipulated genetically to induce models of human disease. Severalmammalian species are also of commercial and agricultural interest.Thus, mammalian genes, multi-gene loci, and genomes are possible targetpolynucleotides for automated nucleic acid-guided nuclease editing inheterologous cells.

The mouse (Mus musculus) genome is approximately 2,700 Mb is size,contains twenty pairs of chromosomes, and approximately 20,000-25,000protein-coding genes (Mouse Genome Sequencing Consortium, et al.,Nature, 420(6915):520-562 (2002)). The chimpanzee (Pan troglodytes)genome is approximately 2,900 Mb in size, contains, 24 pairs ofchromosomes, and approximately 20,000-25,000 protein-coding genes (TheChimpanzee Sequencing and Analysis Consortium, et al., Nature,437(7055):69-87 (2005)). The domestic dog (Canus lupus familiaris)genome is approximately 2,500 Mb in size, contains 39 pairs ofchromosomes, and approximately 20,000-25,000 protein-coding genes(Lindblad-Toh et al., Nature, 438(7069):803-819 (2005). The domestic pig(Sus scrofa) genome is approximately 2,600 Mb in size, contains 19 pairsof chromosomes, and approximately 20,000-25,000 protein-coding genes(Groenen et al., Nature, 491(7424):393-398 (2012)). The domestic cow(Bos Taurus) genome is approximately 2,900 Mb in size, contains 30 pairsof chromosomes, and approximately 22,000 protein-coding genes (Liu etal., BMC Genomics, 10:1-11 (2009)).

Mammalian genomic DNA may be isolated according to standard techniquesand target polynucleotides may be amplified or isolated and introducedto shuttle vectors according to techniques described below. Choice ofshuttle vector depends on target size, destination, and application.

Isolating and characterizing the structure or function of human genesand the human genome are useful research tools for elucidating the causeof human disease and as a guide for developing therapies. Thus, humangenes, multi-gene loci, and the human genome are possible targetpolynucleotides for automated nucleic acid-guided nuclease editing inheterologous cells. The human genome is approximately 3,200 Mb in size,contains 23 pairs of chromosomes, and approximately 20,000-25,000protein-coding genes (International Human Genome Sequencing Consortium,et al., Nature, 431(7011):931-945 (2004)).

Human genomic DNA may be isolated according to standard techniques andtarget polynucleotides may be amplified or isolated and introduced toshuttle vectors according to techniques described below. Choice ofshuttle vector depends on target size, destination, and application.

For all the organisms described herein of which genes, multi-gene loci,and genomes are possible target polynucleotides of automated nucleicacid-guided nuclease editing in heterologous cells, the mitochondrialgenome, when applicable, is also a possible target polynucleotide.

Shuttle Vectors

It may be necessary to use various shuttle vectors in order to introduceexogenous polynucleotides from a source cell or genome into heterologousediting cells for automated nucleic acid-guided nuclease editing.Selection of an appropriate shuttle vector depends on the size of thepayload, the source genome, the source locus, the heterologous cells inwhich automated editing takes place, and the eventual destination ofedited sequences. The following paragraphs discuss example shuttlevectors as well as some methods and considerations for use thereof inshuttling exogenous polynucleotides from the source to the heterologousediting cells in which automated editing occurs.

In some implementations, a bacterial plasmid may be used as a shuttlevector to introduce exogenous polynucleotides from a source locus (orloci) into heterologous editing cells for automated nucleic acid-guidednuclease editing. Plasmids are closed circular DNA molecules that arecomparatively small relative to bacterial genomes and are geneticelements that may exist outside of the genomic chromosome and canreplicate autonomously. Bacterial plasmids typically comprise certainfeatures—natural or engineered—that make them useful for introducingexogenous nucleic acids into cells (Casali & Preston, Plasmid Vectors(Humana Press) (2003)). For example, useful bacterial plasmids comprisean origin of replication to propagate and maintain plasmid copy numberthroughout host cell division, such as the pMB1, pBR322, ColE1, R6K,p15A, pSC101 ColE1, F1, or pUC origin of replication, among others.

Useful bacterial plasmid shuttle vectors also typically comprise anantibiotic resistance gene for use as a selectable marker, allowing fordetection of plasmid-containing cells when grown in or on a selectivegrowth medium. For example, a bacterial plasmid may contain a geneconferring resistance to kanamycin, spectinomycin, streptomycin,ampicillin, carbenicillin, bleomycin, erythromycin, polymyxin B,tetracycline, or chloramphenicol, among others.

Useful bacterial plasmid shuttle vectors may also comprise a multiplecloning site or “polylinker” region. A polylinker is a segment ofnucleotide sequence within the plasmid which contains many—e.g., up to˜20—restriction sites for recognition by restriction endonucleases. Therestrictions sites within the polylinker may be unique, i.e. only occuronce within the sequence of the circular plasmid. Inserts may beconveniently introduced to the shuttle vector by means of the uniquerestriction site and conventional “sticky end” cloning, discussed supra.

Bacterial plasmid shuttle vectors may accommodate an inserted targetpolynucleotide of size 1-100 nucleotides (nt), 50-500 nt, 100-1000 nt,500-5000 nt, 1000-10,000 nt, or 5000-50,000 nt.

In other implementations, yeast plasmids may be used as shuttle vectorsto introduce exogenous polynucleotides from a source cell intoheterologous editing cells for automated nucleic acid-guided nucleaseediting. Similar to bacterial plasmids, yeast plasmids are closedcircular DNA molecules that are comparatively small relative to thelinear chromosomes of the yeast genome. As with bacterial plasmids,there are certain features that are included in yeast plasmids(naturally-occurring or engineered) that make yeast plasmids useful forintroducing exogenous polynucleotides into heterologous editing cells.

In some implementations a yeast centromeric plasmid may be used as ashuttle vector. Yeast centromeric plasmids contain two requisitefeatures: autonomously replicating sequences (ARS) and centromeric (CEN)sequences. Yeast centromeric plasmids exploit the host cell's endogenousreplication and chromosome segregation machinery to persist in yeastcells like mini-chromosomes. The ARS feature functions similarly to thebacterial origin of replication described above and allows forindependent propagation of the plasmid in cells. The CEN sequence is theattachment point for kinetochore complexes and allows for faithfulsegregation of copies of the plasmid to daughter cells during mitosis.Both the ARS and CEN sequences are required for stable maintenance andcorrect distribution of a yeast centromeric plasmid during celldivision. Yeast CEN plasmids are maintained at about 1 copy per cell perhaploid genome, when averaged across the cell populations, althoughplasmid copy number within individual cells may vary (Gnugge and Rudolf,Yeast, 34(4):205-221 (2017)).

In addition to the ARS and CEN sequences, a yeast centromeric plasmidmay contain a polylinker region (discussed supra) and may contain one ormore selectable markers. Yeast shuttle vectors may be maintained inculture by auxotrophic selection, markers for which may include, but arenot limited to, URA3, LEU2, HISS, TRP1, ADE2, LYS2, or MET15.Autoselection systems may include, but are not limited to, URA3, FBAI,POT/TPI, or CDCx. Dominant selectable marker genes may include, but arenot limited to, kan, hph, nat, pat, ble, amdSYM, or tk.

Yeast plasmid shuttle vectors may accommodate an inserted targetpolynucleotide of size 1-100 nucleotides (nt), 50-500 nt, 100-1000 nt,500-5000 nt, 1000-10,000 nt, or 5000-50,000 nt.

In some implementations, a plasmid shuttle vector may be suited for usein both E. coli cells and yeast cells, having properties of bothbacterial and yeast plasmids. In some implementations target sequencesmay be constructed, manipulated, analyzed, cloned, expanded, propagated,edited, transformed into, and isolated out of both bacterial cells andyeast cells. Such a shuttle vector may require a combination of thefollowing (all of which are discussed infra): bacterial origin ofreplication; marker for selection in bacterial cells; yeast ARS; yeastCEN sequence; marker for selection in yeast cells; and a polylinkersequence.

In some implementations—in particular for isolating and deliveringlarger genomic loci or entire genomes to heterologous editing cells forautomated nucleic acid-guided nuclease editing—non-plasmid shuttlevectors may be more appropriate. The paragraphs below discuss variousclasses of artificial chromosomes as well as some methods andconsiderations for use in shuttling exogenous polynucleotides from asource cell or genome to the heterologous editing cells in whichautomated editing occurs.

Bacterial artificial chromosomes (BACs) are DNA constructs capable ofaccommodating larger inserts than plasmids. Similar circular cloningshuttle vectors called PACs are derived from the DNA of P1bacteriophage. BACs and PACs have the advantage of being able to capturelarge genomic segments up to approximately 350 kilobases, therebyfacilitating the cloning of entire genes including noncoding regions andregulatory elements. Hallmark features of BACs and PACs include, but arenot limited to, a P1 or F origin of replication; an antibioticresistance gene which may be one of several genes conferring resistanceto kanamycin, spectinomycin, streptomycin, ampicillin, carbenicillin,bleomycin, erythromycin, polymyxin B, tetracycline, or chloramphenicol,among others; a parA and/or parB sequence for partitioning F plasmid DNAto daughter cells during cell division; and a polylinker region. Asuitable BAC or PAC vector backbone may accommodate an entire gene orother large locus, or a library of inserts generated from a fractionatedgenome. Methods and considerations for introducing large inserts intoBAC and PAC shuttle vectors are discussed supra.

BAC and PAC shuttle vectors may accommodate an inserted targetpolynucleotide of size 10,000-50,000 nucleotides (nt), 30,000-100,000nt, 50,000-300,000 nt, or 100,000-500,000 nt.

Similar to BACs, yeast artificial chromosomes (YACs) may be used asshuttle vectors to accommodate relatively large payloads ofpolynucleotides. In some implementations YACs may be preferable to BACsfor the cloning and shuttling of exogenous polynucleotides intoheterologous editing cells for automated nucleic acid-guided nucleaseediting. Hallmark features of YACs may include, but are not limited to,an ARS sequence (discussed supra) to allow the YAC to replicateautonomously and extrachromosomally; CEN sequences (discussed supra) toconfer mitotic stability and allow for faithful segregation andmaintenance of copy number during cell division; a selectable markerwhich may include, but is not limited to, URA3, LEU2, HISS, TRP1, ADE2,LYS2, or MET15, an autoselection system URA3, FBAI, POT/TPI, or CDCx, ora dominant selectable marker gene may including, kan, hph, nat, pat,ble, amdSYM, or tk. YACs may accommodate relatively large polynucleotideinsert sizes up to 3000 kb in length (Dunnen, et al., Hum Mol Genet,1(1):19-28 (1992)).

YAC shuttle vectors may accommodate an inserted target polynucleotide ofsize 10,000-50,000 nucleotides (nt), 30,000-100,000 nt, 50,000-300,000nt, 100,000-1,000,000 nt, or 300,000-3,000,000 nt.

In some implementations, it may be desirable to use an artificialchromosome shuttle vector that shares features of both bacterial andyeast artificial chromosomes; for example, when a genomic locus isintroduced into a YAC by transformation-associated recombinational (TAR)cloning in yeast (discussed infra) but propagation and rapid large-scaleDNA preparation in bacteria is desired. Such an artificial chromosomemay contain a yeast CEN sequence, yeast ARS sequence, marker forselection in yeast, marker for selection in bacteria, and bacterialorigin of replication, along with the insert of an exogenouspolynucleotide that is the target of automated nucleic acid-guidednuclease editing (Bhargava, et al., Genomics, 62(2):285-288 (1999)). Insome implementations, it may be desirable to retrofit a targetpolynucleotide-carrying YAC into a BAC, e.g. for the purposes ofsimplified preparation of large quantities of shuttle vector DNA, fordownstream transfection into mammalian cells, or for performingautomated nucleic acid-guided nuclease editing of target polynucleotidescloned in YACs in bacterial editing cells rather than yeast editingcells. Retrofitting may be accomplished by transforming theYAC-containing yeast strain with a restriction-digestion linearizedretrofitting plasmid. The retrofitting plasmid may contain a yeastselectable marker, a mammalian selectable marker, the F-factor origin ofreplication, and an antibiotic resistance gene. Recombination betweenthe retrofitting vector and the YAC is mediated by two short targetingsequences with homology to the YAC. After recombination the resultingBAC contains the target polynucleotide previously carried on the YAC(see US Pub. No. 2004/0245317).

The ability to generate fully-functional mammalian artificialchromosomes represents a powerful system for cell-based correction ofgenetic disorders, production of recombinant proteins in transgenicanimals, analysis of regulation and expression of large human genes in avariety of cell types as well as in animal models of human disease,studies of meiosis and chromosome structure, directing celldifferentiation and dedifferentiation, formation of induced pluripotentstem cells, and manipulation of large DNA elements such as but notlimited to chromosome arm exchange onto the synthetic chromosome orincorporation of multiple large DNA elements onto the artificialchromosome. Fully-functional mammalian artificial chromosomes offerseveral advantages over viral-based delivery systems including increasedpayload size, the fact that extrachromosomal maintenance avoidspotential host-cell disruption, avoidance of transcriptional silencingof introduced genes and possible immunological complications, andmammalian artificial chromosomes can be derived from and tailored to thespecies into which the artificial chromosome is to be inserted, e.g.mouse, human.

The synthetic chromosome shuttle vectors and the methods and automatedmulti-module cell editing instruments are applicable to allcurrently-employed methods of artificial chromosome production,including the “top down”, “bottom up”, engineering of minichromosomes,and induced de novo chromosome generation methods used in the art. The“bottom up” approach of artificial chromosome formation relies oncell-mediated de novo chromosome formation following transfection of apermissive cell line with cloned a-satellite sequences, which comprisetypical host cell-appropriate centromeres and selectable marker gene(s),with or without telomeric and genomic DNA. (For protocols and a detaileddescription of these methods see, e.g., Harrington, et al., Nat. Genet.,15:345-55 (1997); Ikeno, et al., Nat. Biotechnol., 16:431-39 (1998);Masumoto, et al., Chromosoma, 107:406-16 (1998), Ebersole, et al., Hum.Mol. Gene., 9:1623-31 (2000); Henning, et al., PNAS USA, 96:592-97(1999); Grimes, et al., EMBO Rep. 2:910-14 (2001); Mejia, et al.,Genomics, 79:297-304 (2002); and Grimes, et al., Mol. Ther., 5:798-805(2002).) Both synthetic and naturally-occurring a-satellite arrays,cloned into yeast artificial chromosomes, bacterial artificialchromosomes or P1-derived artificial chromosome shuttle vectors havebeen used in the art for de novo mammalian artificial chromosomeformation. The products of bottom up assembly can be linear or circular,comprise simplified and/or concatemerized input DNA with an a-satelliteDNA based centromere, and typically range between 1 and 10 Mb in size.Bottom up-derived artificial chromosomes also are engineered toincorporate nucleic acid sequences that permit site-specific integrationof target DNA sequence onto the artificial chromosome.

The “top down” approach of producing mammalian artificial chromosomesinvolves sequential rounds of random and/or targeted truncation ofpre-existing chromosome arms to result in a pared down artificialchromosome comprising a centromere, telomeres, and DNA replicationorigins. (For protocols and a detailed description of these methods see,e.g., Heller, et al., PNAS USA, 93:7125-30 (1996); Saffery, et al., PNASUSA, 98:5705-10 (2001); Choo, Trends Mol. Med., 7:235-37 (2001);Barnett, et al., Nuc. Ac. Res., 21:27-36 (1993); Fan, et al., PNAS USA,88:7006-10 (1991); and Katoh, et al., Biochem. Biophys. Res. Commun.,321:280-90 (2004).) “Top down” artificial chromosomes are constructedoptimally to be devoid of naturally-occuring expressed genes and areengineered to contain DNA sequences that permit site-specificintegration of target DNA sequences onto the truncated chromosome,mediated, e.g., by site-specific DNA integrases.

A third method for producing artificial chromosomes known in the art isengineering of naturally occurring minichromosomes. This productionmethod typically involves irradiation-induced fragmentation of achromosome containing a functional, e.g., human neocentromere possessingcentromere function yet lacking a-satellite DNA sequences and engineeredto be devoid of non-essential DNA. (For protocols and a detaileddescription of these methods see, e.g., Auriche, et al., EMBO Rep.2:102-07 (2001); Moralli, et al., Cytogenet. Cell Genet., 94:113-20(2001); and Carine, et a., Somat. Cell Mol. Genet., 15:445-460 (1989).)As with other methods for generating artificial chromosomes, engineeredminichromosomes can be engineered to contain DNA sequences that permitsite-specific integration of target DNA sequences.

The fourth approach for production of artificial chromosomes involvesinduced de novo chromosome generation by targeted amplification ofspecific chromosomal segments. This approach involves large-scaleamplification of pericentromeric/ribosomal DNA regions situated onacrocentric chromosomes. The amplification is triggered byco-transfection of excess DNA specific to the pericentric region ofchromosomes, such as ribosomal RNA, along with DNA sequences that allowfor site-specific integration of target DNA sequences and also a drugselectable marker which integrates into the pericentric regions of thechromosomes. (For protocols and a detailed description of these methodssee, e.g., Csonka, et al., J. Cell Sci 113:3207-16 (2002); Hadlaczky, etal., Curr. Opini. Mol. Ther., 3:125-32 (2001); and Lindenbaum andPerkins, et al., Nuc. Ac. Res., 32(21):e172 (2004).) During thisprocess, targeting to the pericentric regions of acrocentric chromosomeswith co-transfected DNA induces large-scale chromosomal DNAamplification, duplication/activation of centromere sequences, andsubsequent breakage and resolution of dicentric chromosomes resulting ina “break-off” satellite DNA-based synthetic chromosome containingmultiple site-specific integration sites.

In some embodiments, the cells used to produce the artificial chromosomemay be the heterologous editing cell; that is, the heterologous editingcell can be used—and preferably is used—for both synthetic chromosomeconstruction and editing. Alternatively, the cells to produce theartificial chromosome (and, in some embodiments, to edit the artificialchromosomes) can be cells that naturally occur in a subject (humanpatient, animal or plant) in which the genes or regulatory sequencesfrom the artificial chromosome will ultimately be expressed. Such cellscan be primary-culture cell lines established for the purpose ofartificial chromosome production specific for an individual. In otherembodiments, the cells to produce the artificial chromosome and, in someembodiments, to edit the artificial chromosomes) are from an establishedcell line. A wide variety of cell lines for tissue culture are known inthe art. Examples of cell lines include but are not limited to humancells lines such as 293-T (embryonic kidney), 721 (melanoma), A2780(ovary), A172 (glioblastoma), A253 (carcinoma), A431 (epithelium), A549(carcinoma), BCP-1 (lymphoma), BEAS-2B (lung), BR 293 (breast), BxPC3(pancreatic cancinoma), Cal-27 (tongue), COR-L23 (lung), COV-434(ovary), CML T1 (leukemia), DUI45 (prostate), DuCaP (prostate), FM3(lymph node), H1299 (lung), H69 (lung), HCA2 (fibroblast), HEK0293(embryonic kidney), HeLa (cervix), HL-60 (myeloblast), HMEC(epithelium), HT-29 (colon), HUVEC (umbilical vein epithelium), Jurkat(T cell leukemia), JY (lymphoblastoid), K562 (lymphoblastoid), KBM-7(lymphoblastoid), Ku812 (lymphoblastoid), KCL22 (lymphoblastoid), KGI(lymphoblastoid), KYO1 (lymphoblastoid), LNCap (prostate), Ma-Mel(melanoma), MCF-7 (mammary gland), MDF-10A (mammary gland), MDA-MB-231,-468 and -435 (breast), MG63 (osteosarcoma), MOR/0.2R (lung), MONO-MAC6(white blood cells), MRCS (lung), MSU1.1 (fibroblast), NCI-H69 (lung),NALM-1 (peripheral blood), NW-145 (melanoma), OPCN/OPCT (prostate), Peer(leukemia), Raji (B lymphoma), Saos-2 (osteosarcoma), Sf21 (ovary), Sf9(ovary), SiHa (cervical cancer), SKBR3 (breast carcinoma), SKOV-2 (ovarycarcinoma), T-47D (mammary gland), T84 (lung), U373 (glioblastoma), U87(glioblastoma), U937 (lymphoma), VCaP (prostate), WM39 (skin), WT-49(lymphoblastoid), and YAR (B cell). Rodent cell lines of interestinclude but are not limited to 3T3 (mouse fibroblast), 4T1 (mousemammary), 9L (rat glioblastoma), A20 (mouse lymphoma), ALC (mouse bonemarrow), B16 (mouse melanoma), B35 (rat neuroblastoma), bEnd.3 (mousebrain), C2C12 (mouse myoblast), C6 (rat glioma), CGR8 (mouse embryonic),CT26 (mouse carcinoma), E14Tg2a (mouse embryo), EL4 mouse leukemia),EMT6/AR1 (mouse mammary), Hepalcic7 (mouse hepatoma), J558L (mousemyeloma), MC-38 (mouse adenocarcinoma), MTD-1A (mouse epithelium), RBL(rat leukemia), RenCa (mouse carcinoma), X63 (mouse lymphoma), YAC-1(mouse Be cell), BHK-1 (hamster kidney), and CHO (hamster ovary). Plantcell lines of use include but are not limited to BY-2, Xan-1, GV7, GF11,GT16, TBY-AtRER1B, 3n-3, and G89 (tobacco); VR, VW, and YU-1 (grape);PAR, PAP, and PAW (pokeweed); Spi-WT, Spi-1-1, and Spi12F (spinach);PSB, PSW and PSG (sesame); A.per, A.pas, A.plo (asparagus); Pn and Pb(bamboo); and DG330 (soybean); embryonic cell lines; pluripotent celllines; adult derived stem cells; reprogrammed cell lines; generic animalcell lines of any species or broadly embryonic or reprogrammed cells;zebra fish cell lines; primary dog cells; primary horse cells; chickenDT40 cells; dog cell lines; cat cell lines; patient cell lines; and, insome preferred embodiments, the HT1080 human cell line is utilized.Potential cells of use include any living cell, but those fromeukaryotes are specifically contemplated. These cell lines and othersare available from a variety of sources known to those with skill in theart (see, e.g., the American Type Culture Collection (ATCC) (Manassas,Va.)).

In some implementations, it may be desirable to insert the edited targetpolynucleotide carried by a YAC or BAC shuttle vector into a mammalianartificial chromosome. For example, the target nucleotide may be amammalian gene cloned into a BAC or YAC shuttle vector, subjected toautomated nucleic acid-guided editing in heterologous editing cells,with a downstream application including expression in a human cell line.The BAC or YAC shuttle vector may be designed to include a loxPrecombination site, and the mammalian artificial chromosome may bedesigned to include a loxP recombination site. The edited polynucleotideis loaded onto the mammalian artificial chromosome by Cre-loxP mediatedrecombination. The YAC or BAC may be co-transfected with aCre-recombinase expression vector into mammalian cells carrying themammalian artificial chromosome. The product of this recombination maybe a mammalian artificial chromosome containing the edited targetpolynucleotide.

FIG. 1 is a process diagram showing the various options or pathways forselecting, building and editing shuttle vectors. The source targetpolynucleotides may be, as described above, e.g., an open reading framecoding sequence, several to many genes in a biochemical pathway, one ormore noncoding regions, a large genomic locus, or an entire genome. Thesource cell may be bacterial cells, fungal cells, non-model organismcells, plant cells, and mammalian cells, including human cells. Also asdescribed above, the shuttle vector itself can be, e.g., a bacterialplasmid, a viral vector, a yeast plasmid, a YAC, a BAC, hybrids ofthese, or artificial chromosomes, including mammalian artificialchromosomes. The construction of the vector and artificial chromosomesand the cloning of the source target polynucleotide can be accomplishedby various methods known in the art, such as PCR amplification andrestriction cloning, restriction fragmenting of an entire genome andcloning into a vector backbone to produce a library,Transformation-Associated Recombination (TAR) cloning, and othercloning, construction, and synthesis methods. As with the source cell,the heterologous editing cells may be of bacterial, fungal, plant,mammalian, or human origin. The edits of the source targetpolynucleotide can be virtually any type of edit, including saturationmutagenesis, knockouts, loss of function mutations, gain of functionchanges, coding variations (including codon changes, and the addition orremoval of stop or start codons), as well as changes to regulatory andother noncoding regions. Finally, the ultimate destination of the editedshuttle vector may be back to the source cell, to another, different,vector, or to yet another bacterial, fungal, plant, mammalian, or humancell.

Cloning Strategies for Introducing Exogenous Genetic Material to ShuttleVectors

The means by which target polynucleotides are introduced to a shuttlevector depend on the choice of shuttle vector, source locus of thepolynucleotide, size of the insert, and for the embodiments disclosedherein, the desired edits of the target polynucleotide and eventualdestination of the edited target polynucleotide. The followingparagraphs discuss strategies for cloning target polynucleotides fromthe source cells, introducing the target polynucleotides to shuttlevectors, as well as some exemplary methods and considerations for thecloning step of shuttling exogenous polynucleotides from the sourcecells to the heterologous editing cells in which automated editingoccurs.

In some implementations, target polynucleotides may be amplified bypolymerase chain reaction (PCR) using oligonucleotide primerscomplementary to sequences flanking the target polynucleotide. PCRprimers may be designed in such a way that the resulting ampliconscontain restriction sites for recognition by restriction endonucleases.Amplicons may then be digested by restriction endonucleases and ligatedinto a, e.g., plasmid shuttle vector at the polylinker region byconventional “sticky end” cloning. The resulting shuttle vector may beintroduced to heterologous editing cells for automated nucleicacid-guided nuclease editing.

Alternatively, in some implementations, target polynucleotides may beamplified by PCR as described above, but with PCR primers designed suchthat the resulting amplicons contain homology arms of size 10-500nucleotides at their 5′ and 3′ ends. These arms may be homologous to asequence in the shuttle vector such that after linearizing the shuttlevector by restriction enzyme digestion the arms may mediate in vitroisothermic assembly (see, e.g., U.S. Pat. No. 7,776,532). This methodhas the advantages of 1) not requiring restriction sites in either thetarget polynucleotide amplicon or in the shuttle vector backbone, and 2)scarless incorporation of the target polynucleotide insert into theshuttle vector.

In yet other implementations, target polynucleotides may be commerciallysynthesized. The target polynucleotide may be synthesized in acommercially-available vector and subcloned into the desired shuttlevector by conventional methods, including but not limited to restrictionenzyme “sticky end” cloning and in vitro isothermic assembly.Alternatively, the shuttle vector may be commercially synthesized incircular plasmid form already containing the target polynucleotide.

For implementations when the target “polynucleotide” is an entiregenome, a genomic library of shuttle vectors may be constructed. Totalgenomic DNA may be fractionated by restriction enzyme digest, e.g. BamHIor EcoRI, size selected by one of various standard methods, e.g. sucrosegradient or gel electrophoresis, and ligated into a linearized BAC orYAC vector backbone (Burke & Olson, Methods Enzymol. 194(c):251-270(1991); and Foote & Denny, Curr Protoc Hum Genet. (2002)), yielding aheterogenous mix of shuttle vectors containing target polynucleotideinserts representing the entire source genome. The library of shuttlevectors may then be introduced to heterologous editing cells forautomated nucleic acid-guided nuclease editing.

In some implementations a target polynucleotide may already be clonedfrom a source and exist in a vector or library. In this case the targetpolynucleotide may be subcloned by restriction enzyme digest, amplifiedby PCR, or other standard techniques, and introduced to a desiredshuttle vector by one of several techniques e.g. restriction enzyme“sticky end” cloning, in vitro isothermal assembly, or in vivorecombination in yeast. The resulting shuttle vector may then beintroduced to heterologous editing cells for automated nucleicacid-guided nuclease editing.

In some implementations, when the target polynucleotide is an entiregene or large multi-gene locus, the target polynucleotide may beintroduced to the shuttle vector by transformation-associatedrecombinational (TAR) cloning in yeast. TAR cloning is a useful methodfor capturing relatively large genomic loci in YACs and allows entiregenes and large chromosomal regions to be selectively and accuratelyisolated from total genomic DNA by in vivo recombination in yeast(Kouprina, et al., Nat Rev Genet., 7:805-812 (2006); Kouprina, et al.,Curr Protoc Hum Genet., Chapter 5 Unit 5.17 (2006)). Advantages of TARcloning include 1) specificity in targeting genomic loci (as opposed torandom library generation), 2) accuracy, and 3) the capacity to clonelarge inserts. TAR cloning vectors may comprise a CEN sequence, yeastselectable marker, yeast telomeric sequences, and at both ends twogene-specific “hooks,” which are two regions of homology that flank thesource genomic target polynucleotide (see U.S. Pat. No. 6,391,642).These hooks mediate recombination between the TAR cloning vector andtarget genomic DNA—which are co-transformed into the heterologousediting cells—resulting in a circular YAC containing the genomic targetpolynucleotide insert. TAR cloning is particularly useful for cloningmammalian target loci, as the method depends on the presence of anARS-like sequence in the cloned insert for the resulting YAC toreplicate faithfully in yeast. ARS-like elements are abundant inmammalian genomes, occurring at a frequency of approximately 20-40 kb.TAR cloning vectors may also have properties of BACs or may beretrofitted after cloning to have properties of BACs and may be shuttledbetween bacteria and yeast, for example, when a genomic locus isintroduced into a YAC by TAR cloning in yeast but propagation and rapidlarge-scale DNA preparation in bacteria is desired. Such shuttle vectorsproduced by TAR cloning may be edited in either yeast or bacteria cells.

Nucleic Acid-Guided Cell Editing Generally

Various shuttle vectors comprising the desired target polynucleotidesdescribed herein (e.g., gene(s), coding sequences, multi-gene loci,regulatory elements, genomes) are edited by nucleic acid-guided editingmethods, modules, instruments and systems in which nucleic acid-guidednucleases (e.g., RNA-guided nucleases) are used to edit specific targetregions in the shuttle vector(s). A nucleic acid-guided nucleasecomplexed with an appropriate synthetic guide nucleic acid in a cell cancut the shuttle vector at a desired location. The guide nucleic acidhelps the nucleic acid-guided nuclease recognize and cut a specifictarget sequence in the shuttle vector. By manipulating the nucleotidesequence of the guide nucleic acid, the nucleic acid-guided nuclease maybe programmed to target any DNA sequence for cleavage as long as anappropriate protospacer adjacent motif (PAM) is nearby.

The components for nucleic acid-guided editing may be delivered toheterologous editing cells in various ways and in various combinations.For example, the nuclease itself may be delivered to a cell comprising ashuttle vector as a polypeptide; alternatively, a polynucleotidesequence encoding the nuclease(s) is transformed or transfected into thecells comprising the shuttle vector to be edited. The polynucleotidesequence encoding the nuclease may be codon optimized for expression inparticular cells, such as eukaryotic cells, including mammalian cells.Eukaryotic cells can be yeast, fungi, algae, plant, animal, or humancells. Eukaryotic cells may be those of or derived from a particularorganism, such as a mammal, including but not limited to human, mouse,rat, rabbit, dog, or non-human mammals including non-human primates. Thechoice of the nuclease to be employed depends on many factors, such aswhat type of edit is to be made in the target sequence and whether anappropriate PAM is located close to the desired target sequence. Thenuclease may be encoded by a DNA sequence on a vector (e.g., an enginevector) and be under the control of a constitutive or induciblepromoter. In some embodiments, the sequence encoding the nuclease isunder the control of an inducible promoter, and the inducible promotermay be separate from but the same as an inducible promoter controllingtranscription of the guide nucleic acid; that is, a separate induciblepromoter may drive the transcription of the nuclease and guide nucleicacid sequences but the two inducible promoters may be the same type ofinducible promoter (e.g., both are pL promoters). Alternatively, theinducible promoter controlling expression of the nuclease may bedifferent from the inducible promoter controlling transcription of theguide nucleic acid; that is, e.g., the nuclease may be under the controlof the pBAD inducible promoter, and the guide nucleic acid may be underthe control of the pL inducible promoter. In yet another example, thecoding sequence for the nuclease may be under the control of aninducible promoter and the transcription sequence for the guide nucleicacid may be under the control of a constitutive promoter.

In general, a guide nucleic acid (e.g., gRNA) complexes with acompatible nucleic acid-guided nuclease and can then hybridize with atarget sequence in the shuttle vector, thereby directing the nuclease tothe target sequence. In certain aspects, a CRISPR editing system may usetwo separate guide nucleic acid molecules that combine to function as aguide nucleic acid, e.g., a CRISPR RNA (crRNA) and trans-activatingCRISPR RNA (tracrRNA). In other aspects, the guide nucleic acid may be asingle guide nucleic acid that includes both the crRNA and tracrRNAsequences. A guide nucleic acid can be DNA or RNA; alternatively, aguide nucleic acid may comprise both DNA and RNA. In some embodiments, aguide nucleic acid may comprise modified or non-naturally occurringnucleotides. In cases where the guide nucleic acid comprises RNA, thegRNA may be encoded by a DNA sequence on a polynucleotide molecule suchas a plasmid, linear construct, or the coding sequence may reside withinan editing cassette and is under the control of a constitutive promoter,or, in some embodiments, an inducible promoter as described below.

A guide nucleic acid comprises a guide sequence, where the guidesequence is a polynucleotide sequence having sufficient complementaritywith a target sequence to hybridize with the target sequence and directsequence-specific binding of a complexed nucleic acid-guided nuclease tothe target sequence. The degree of complementarity between a guidesequence and the corresponding target sequence, when optimally alignedusing a suitable alignment algorithm, is about or more than about 50%,60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment maybe determined with the use of any suitable algorithm for aligningsequences. In some embodiments, a guide sequence is about or more thanabout 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length.In some embodiments, a guide sequence is less than about 75, 50, 45, 40,35, 30, 25, 20 nucleotides in length. Preferably the guide sequence is10-30 or 15-20 nucleotides long, or 15, 16, 17, 18, 19, or 20nucleotides in length.

In the present methods and instruments, the guide nucleic acid typicallyis provided as a sequence to be expressed from a plasmid or vector andcomprises both the guide sequence and the scaffold sequence as a singletranscript under the control of a promoter, and in some embodiments, aninducible promoter. The guide nucleic acid can be engineered to target adesired target sequence in a shuttle vector by altering the guidesequence so that the guide sequence is complementary to a desired targetsequence, thereby allowing hybridization between the guide sequence andthe target sequence. In general, to generate an edit in the targetsequence, the gRNA/nuclease complex binds to a target sequence asdetermined by the guide RNA, and the nuclease recognizes a protospaceradjacent motif (PAM) sequence adjacent to the target sequence. Herein,the target sequence is contained within a shuttle vector that isexogenous to a destination cell. A target sequence can be a sequenceencoding a gene product (e.g., a protein) or a non-coding sequence(e.g., a regulatory polynucleotide, an intron, a PAM, or “junk” DNA).

The guide nucleic acid may be part of an editing cassette that encodesthe donor nucleic acid. Alternatively, the guide nucleic acid may not bepart of the editing cassette and instead may be encoded on the engine orediting vector backbone. For example, a sequence coding for a guidenucleic acid can be assembled or inserted into a vector backbone first,followed by insertion of the donor nucleic acid in, e.g., the editingcassette. In other cases, the donor nucleic acid in, e.g., an editingcassette can be inserted or assembled into a vector backbone first,followed by insertion of the sequence coding for the guide nucleic acid.In yet other cases, the sequence encoding the guide nucleic acid and thedonor nucleic acid (inserted, for example, in an editing cassette) aresimultaneously but separately inserted or assembled into a shuttlevector. In yet other embodiments, the sequence encoding the guidenucleic acid and the sequence encoding the donor nucleic acid are bothincluded in the editing cassette. Methods and compositions for designingand synthesizing editing cassettes are described in U.S. Pat. Nos.10,240,167; 10,266,849; 9,982,278; 10,351,877; 10,364,442; and10,435,715; and U.S. Ser. No. 16/275,465, filed 14 Feb. 2019, all ofwhich are incorporated by reference herein.

The target sequence is associated with a PAM, which is a shortnucleotide sequence recognized by the gRNA/nuclease complex. The precisePAM sequence and length requirements for different nucleic acid-guidednucleases vary; however, PAMs typically are 2-7 base-pair sequencesadjacent or in proximity to the target sequence and, depending on thenuclease, can be 5′ or 3′ to the target sequence. Engineering of thePAM-interacting domain of a nucleic acid-guided nuclease may allow foralteration of PAM specificity, improve target site recognition fidelity,reduce cutting efficiency, decrease target site recognition fidelity, orincrease the versatility of a nucleic acid-guided nuclease. In certainembodiments, the editing of a target sequence both introduces a desiredDNA change to a target sequence, e.g., the desired sequences containedon the shuttle vector within a cell, and removes, mutates, or rendersinactive a proto-spacer mutation (PAM) region in the target sequence.Rendering the PAM at the target sequence inactive precludes additionalediting of the shuttle vector at that target sequence, e.g., uponsubsequent exposure to a nucleic acid-guided nuclease complexed with asynthetic guide nucleic acid in later rounds of editing.

The range of target sequences that nucleic acid-guided nucleases canrecognize is constrained by the need for a specific PAM to be locatednear the desired target sequence. As a result, it often can be difficultto target edits with the precision that is necessary for editing. It hasbeen found that nucleases can recognize some PAMs very well (e.g.,canonical PAMs), and other PAMs less well or poorly (e.g., non-canonicalPAMs).

Another component of the nucleic acid-guided nuclease system is thedonor nucleic acid. In some embodiments, the donor nucleic acid is onthe same polynucleotide (e.g., editing vector or editing cassette) asthe guide nucleic acid and may be (but not necessarily) under thecontrol of the same promoter as the guide nucleic acid (e.g., a singlepromoter driving the transcription of both the guide nucleic acid andthe donor nucleic acid). The donor nucleic acid is designed to serve asa template for homologous recombination with a target sequence nicked orcleaved by the nucleic acid-guided nuclease as a part of thegRNA/nuclease complex. A donor nucleic acid polynucleotide may be of anysuitable length, such as about or more than about 20, 25, 50, 75, 100,150, 200, 500, or 1000 nucleotides in length. In certain preferredaspects, the donor nucleic acid can be provided as an oligonucleotide ofbetween 20-300 nucleotides, more preferably between 50-250 nucleotides.The donor nucleic acid comprises a region that is complementary to aportion of the target sequence (e.g., a homology arm). When optimallyaligned, the donor nucleic acid overlaps with (is complementary to) thetarget sequence by, e.g., about 20, 25, 30, 35, 40, 50, 60, 70, 80, 90or more nucleotides. In many embodiments, the donor nucleic acidcomprises two homology arms (regions complementary to the targetsequence) flanking the mutation or difference between the donor nucleicacid and the target template. The donor nucleic acid comprises at leastone mutation or alteration compared to the target sequence, such as aninsertion, deletion, modification, or any combination thereof comparedto the target sequence.

Often the donor nucleic acid is provided as an editing cassette, whichis inserted into a vector backbone where the vector backbone maycomprise a promoter driving transcription of the gRNA and the codingsequence of the gRNA, or the vector backbone may comprise a promoterdriving the transcription of the gRNA but not the gRNA itself. Moreover,there may be more than one, e.g., two, three, four, or more guidenucleic acid/donor nucleic acid cassettes inserted into an enginevector, where each guide nucleic acid is under the control of separatedifferent promoters, separate like promoters, or where all guide nucleicacid/donor nucleic acid pairs are under the control of a singlepromoter. In some embodiments—such as embodiments where cell selectionis employed—the promoter driving transcription of the gRNA and the donornucleic acid (or driving more than one gRNA/donor nucleic acid pair) isan inducible promoter. Inducible editing is advantageous in thatsingulated or substantially singulated cells can be grown for several tomany cell doublings before editing is initiated, which increases thelikelihood that cells with edits will survive, as the double-strand cutscaused by active editing are largely toxic to the cells. This toxicityresults both in cell death in the edited colonies, as well as a lag ingrowth for the edited cells that do survive but must repair and recoverfollowing editing. However, once the edited cells have a chance torecover, the size of the colonies of the edited cells will eventuallycatch up to the size of the colonies of unedited cells. See, e.g., U.S.Pat. No. 10,550,363, issued 4 Feb. 2020. Further, a guide nucleic acidmay be efficacious directing the edit of more than one donor nucleicacid in an editing cassette; e.g., if the desired edits are close to oneanother in a target sequence.

In addition to the donor nucleic acid, an editing cassette may compriseone or more primer sites. The primer sites can be used to amplify theediting cassette by using oligonucleotide primers; for example, if theprimer sites flank one or more of the other components of the editingcassette.

Also, as described above, the donor nucleic acid may comprise—inaddition to the at least one mutation relative to a target sequence—oneor more PAM sequence alterations that mutate, delete or render inactivethe PAM site in the target sequence. The PAM sequence alteration in thetarget sequence in the shuttle vector renders the PAM site “immune” tothe nucleic acid-guided nuclease and protects the target sequence fromfurther editing in subsequent rounds of editing if the same nuclease isused.

In addition, the editing cassette may comprise a barcode. A barcode is aunique DNA sequence that corresponds to the donor DNA sequence such thatthe barcode can identify the edit made to the corresponding targetsequence. The barcode typically comprises four or more nucleotides. Insome embodiments, the editing cassettes comprise a collection of donornucleic acids representing, e.g., gene-wide or shuttle vector-widelibraries of donor nucleic acids. The library of editing cassettes arecloned into vector backbones where, e.g., each different donor nucleicacid is associated with a different barcode.

Additionally, in some embodiments, an expression vector or cassetteencoding components of the nucleic acid-guided nuclease system furthercomprises one or more nuclear localization sequences (NLSs), such asabout or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs. Insome embodiments, the vectors encoding the editing components compriseNLSs at or near the amino-terminus, NLSs at or near thecarboxy-terminus, or a combination.

The engine and editing vectors comprise control sequences operablylinked to the component sequences to be transcribed. As stated above,the promoters driving transcription of one or more components of thenuclease editing system may be inducible, and an inducible system islikely employed if selection is to be performed. A number of generegulation control systems have been developed for the controlledexpression of genes in plant, and animal cells, including mammaliancells, including the pL promoter (induced by heat inactivation of theCI857 repressor), the pBAD promoter (induced by the addition ofarabinose to the cell growth medium), and the rhamnose induciblepromoter (induced by the addition of rhamnose to the cell growthmedium). Other systems include the tetracycline-controlledtranscriptional activation system (Tet-On/Tet-Off, Clontech, Inc. (PaloAlto, Calif.); Bujard and Gossen, PNAS, 89(12):5547-5551 (1992)), theLac Switch Inducible system (Wyborski et al., Environ Mol Mutagen,28(4):447-58 (1996); DuCoeur et al., Strategies 5(3):70-72 (1992); U.S.Pat. No. 4,833,080), the ecdysone-inducible gene expression system (Noet al., PNAS, 93(8):3346-3351 (1996)), the cumate gene-switch system(Mullick et al., BMC Biotechnology, 6:43 (2006)), and thetamoxifen-inducible gene expression (Zhang et al., Nucleic AcidsResearch, 24:543-548 (1996)) as well as others.

Performing genome editing in live cells entails transforming cells withthe components necessary to perform nucleic acid-guided nucleaseediting. For example, the cells may be transformed simultaneously withseparate engine and editing vectors; the cells may already be expressinga nuclease (e.g., the cells may have already been transformed with anengine vector or the coding sequence for the nuclease may be stablyintegrated into the cellular genome) such that only the editing vectorneeds to be transformed into the cells; or the cells may be transformedwith a single vector comprising all components required to performnucleic acid-guided nuclease genome editing.

A variety of delivery systems can be used to introduce (e.g., transformor transfect) nucleic acid-guided nuclease editing system componentsinto a heterologous editing cell. These delivery systems include the useof yeast systems, lipofection systems, microinjection systems, biolisticsystems, virosomes, liposomes, immunoliposomes, polycations,lipid:nucleic acid conjugates, virions, artificial virions, viralvectors, electroporation, cell permeable peptides, nanoparticles,nanowires, exosomes. Alternatively, molecular trojan horse liposomes maybe used to deliver nucleic acid-guided nuclease components across theblood brain barrier. Of particular interest is the use ofelectroporation, particularly flow-through electroporation (either as astand-alone instrument or as a module in an automated multi-modulesystem) as described in, e.g., U.S. Pat. No. 10,435,713, issued 8 Oct.2019; U.S. Pat. No. 10,443,074, issued 15 Oct. 2019; U.S. Pat. No.10,323,258, issued 18 Jun. 2019; U.S. Pat. No. 10,568,288, issued 17Dec. 2019; and U.S. Pat. No. 10,415,058, issued 17 Sep. 2019.

After the cells are transformed with the components necessary to performnucleic acid-guided nuclease editing, the cells are cultured underconditions that promote editing. For example, if constitutive promotersare used to drive transcription of the nuclease and/or gRNA, thetransformed cells need only be cultured in a typical culture mediumunder typical conditions (e.g., temperature, CO₂ atmosphere, etc.)Alternatively, if editing is inducible—by, e.g., activating induciblepromoters that control transcription of one or more of the componentsneeded for nucleic acid-guided nuclease editing, such as, e.g.,transcription of the gRNA, donor DNA, and nuclease—the cells aresubjected to inducing conditions.

Production of Cell Libraries Comprising Shuttle Vectors Using AutomatedEditing Methods and Instruments

In one aspect, the present disclosure provides automated editing methodsand multi-module cell editing instruments for creating a library ofcells that vary the expression, levels and/or activity of RNAs and/orproteins of interest in cells using various editing strategies, asdescribed herein in more detail. Accordingly, the disclosure is intendedto cover edited cell libraries comprising the shuttle vectors created bythe automated editing methods and multi-module cell editing instrumentsof the disclosure. These cell libraries may have different targetededits, including but not limited to gene knockouts, gene knock-ins,insertions, deletions, single nucleotide edits, short tandem repeatedits, frameshifts, and triplet codon expansion. These edits can bedirected to coding or non-coding regions of the genome and arepreferably rationally designed.

In specific aspects, the cell libraries are created using multiplexedediting of individual cells comprising one or more shuttle vectorswithin a cell population, where multiple cells within a cell populationare edited in a single round of editing, i.e., multiple changes withinthe shuttle vectors of the cells of the cell library are edited in asingle automated operation. The libraries that can be created in asingle multiplexed automated operation can comprise as many as 500edited cells, 1000 edited cells, 2000 edited cells, 5000 edited cells,10,000 edited cells, 50,000 edited cells, 100,000 edited cells, 200,000edited cells, 300,000 edited cells, 400,000 edited cells, 500,000 editedcells, 600,000 edited cells, 700,000 edited cells, 800,000 edited cells,900,000 edited cells, 1,000,000 edited cells, 2,000,000 edited cells,3,000,000 edited cells, 4,000,000 edited cells, 5,000,000 edited cells,6,000,000 edited cells, 7,000,000 edited cells, 8,000,000 edited cells,9,000,000 edited cells, 10,000,000 edited cells or more, and the numberof different types of edits that can be created in a single multiplexedautomated operation can comprise 500, 1,000, 5,000, 10,000, 20,000,25,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000 or up to100,000 edits or more.

In other specific aspects, the cell libraries are created usingrecursive editing of shuttle vectors of individual cells within a cellpopulation, with edits being added to the individual cells in two ormore rounds of editing. The use of recursive editing results in theamalgamation of two or more edits targeting two or more sites in ashuttle vector in individual cells of the library. The libraries thatcan be created in an automated recursive operation can comprise as manyas 500 edited cells, 1000 edited cells, 2000 edited cells, 5000 editedcells, 10,000 edited cells, 50,000 edited cells, 100,000 edited cells,200,000 edited cells, 300,000 edited cells, 400,000 edited cells,500,000 edited cells, 600,000 edited cells, 700,000 edited cells,800,000 edited cells, 900,000 edited cells, 1,000,000 edited cells,2,000,000 edited cells, 3,000,000 edited cells, 4,000,000 edited cells,5,000,000 edited cells, 6,000,000 edited cells, 7,000,000 edited cells,8,000,000 edited cells, 9,000,000 edited cells, 10,000,000 edited cellsor more, and the number of different types of edits that can be createdin recursive multiplexed automated operations can comprise 500, 1,000,5,000, 10,000, 20,000, 25,000, 30,000, 40,000, 50,000, 60,000, 70,000,80,000, 90,000 or up to 100,000 edits or more per round of editing.

In specific aspects, recursive editing can be used to first create acell phenotype, and then later rounds of editing used to reverse thephenotype and/or accelerate other cell properties.

In some aspects, the cell library comprises edits for the creation ofunnatural amino acids in a cell.

In specific aspects, there are provided cell libraries having edits inone or more regulatory elements in the shuttle vectors created using theautomated editing methods and multi-module cell editing instruments ofthe disclosure. The term “regulatory element” refers to nucleic acidmolecules that can influence the transcription and/or translation of anoperably linked coding sequence in a particular environment and/orcontext. This term is intended to include all elements that promote orregulate transcription, and RNA stability including promoters, coreelements required for basic interaction of RNA polymerase andtranscription factors, upstream elements, enhancers, transcriptionfactor binding sites, and response elements. Exemplary regulatoryelements used in eukaryotic cells may include, but are not limited to,promoters, enhancers, insulators, splicing signals and polyadenylationsignals.

Preferably, the edited cell library includes rationally-designed editsthat are designed based on predictions of protein structure, expressionand/or activity in a particular cell type. For example, rational designmay be based on a system-wide biophysical model of genome editing with aparticular nuclease and gene regulation to predict how different editingparameters including nuclease expression and/or binding, growthconditions, and other experimental conditions collectively control thedynamics of nuclease editing. See, e.g., Farasat and Salis, PLoS ComputBiol., 29:12(1):e1004724 (2016).

In one aspect, there is provided creation of a library of edited cellswith various rationally-designed regulatory sequences created usingautomated editing modules, instrumentation, systems and methods. In oneexample, the edited cell library includes eukaryotic sequences createdusing a set of constitutive and/or inducible promoters, enhancersequences, operator sequences, and/or different Kozak sequences forexpression of proteins of interest.

In some aspects, there are provided cell libraries including cells withrationally-designed edits comprising one or more classes of edits insequences of interest across the shuttle vectors.

Importantly, in certain aspects the cell libraries may comprise editsusing randomized sequences, e.g., randomized promoter sequences, toreduce similarity between expression of one or more proteins inindividual cells within the library. Additionally, the promoters in thecell library can be constitutive, inducible or both to enable strongand/or titratable expression.

In other aspects, the present disclosure provides automated editingmethods and multi-module cell editing instruments for creating a libraryof cells comprising edits to identify optimum expression of a selectedgene target. For example, production of biochemicals through metabolicengineering often requires the expression of pathway enzymes, and thebest production yields are not always achieved by the highest amount ofthe target pathway enzymes in the cell, but rather by fine-tuning of theexpression levels of the individual enzymes and related regulatoryproteins and/or pathways. Similarly, expression levels of heterologousproteins sometimes can be experimentally adjusted for optimal yields.

One way that transcription impacts gene expression levels is through therate of Pol II initiation, which can be modulated by combinations ofpromoter or enhancer strength and trans-activating factors (see, e.g.,Kadonaga, et al., Cell, 116(2):247-57 (2004)). In eukaryotes, elongationrate may also determine gene expression patterns by influencingalternative splicing (Cramer, et al., PNAS USA, 94(21):11456-60 (1997)).Failed termination on a gene can impair the expression of downstreamgenes by reducing the accessibility of the promoter to Pol II (Greger,et al., PNAS USA, 97(15):8415-20 (2000)). This process, known astranscriptional interference, is particularly relevant in lowereukaryotes, as they often have closely spaced genes.

In some embodiments there are provided methods for optimizing cellulargene transcription. Gene transcription is the result of several distinctbiological phenomena, including transcriptional initiation (RNAprecruitment and transcriptional complex formation), elongation (strandsynthesis/extension), and transcriptional termination (RNAp detachmentand termination).

Site Directed Mutagenesis

Cell libraries can be created via site-directed mutagenesis using theautomated editing methods, modules, instruments, and systems, i.e., whenthe amino acid sequence of a protein or other genomic feature preferablyis to be altered by deliberately and precisely by mutating the proteinor genomic feature. These cell libraries can be useful for variouspurposes, e.g., for determining protein function within cells, foridentifying of enzymatic active sites within cells, and for designingnovel proteins. For example, site-directed mutagenesis can be used in amultiplexed fashion to exchange a single amino acid in the sequence of aprotein for another amino acid with different chemical properties. Thisallows one to determine the effect of a rationally-designed orrandomly-generated mutation in individual cells within a cellpopulation. See, e.g., Berg, et al. Biochemistry, Sixth Ed. (New York:W.H. Freeman and Company) (2007).

In another example, edits can be made to individual cells within a celllibrary to substitute amino acids in binding sites, such as substitutionof one or more amino acids in a protein binding site for interactionwithin a protein complex or substitution of one or more amino acids inenzymatic pockets that can accommodate a cofactor or ligand. This classof edits allows the creation of specific manipulations to measurecertain properties of one or more proteins, including interaction withother cofactors, ligands, etc. within a protein complex.

In yet another examples, various edit types can be made to individualcells within a cell library using site specific mutagenesis for studyingexpression quantitative trait loci (eQTLs). An eQTL is a locus thatexplains a fraction of the genetic variance of a gene expressionphenotype. The libraries are useful to evaluate and link eQTLs to actualdiseased states.

In specific aspects, the edits introduced into the shuttle vectors tocreate the cell libraries may be created using rational design based onknown or predicted structures of proteins. See, e.g., Chronopoulou andLabrou, Curr Protoc Protein Sci.; Chapter 26:Unit 26.6 (2011). Suchsite-directed mutagenesis can provide individual cells within a librarywith one or more site-directed edits, and preferably two or moresite-directed edits (e.g., combinatorial edits) within a cellpopulation.

In other aspects, cell libraries of the disclosure are created usingsite-directed codon mutation “scanning” of all or substantially all ofthe codons in the coding region of a gene. In this fashion, individualedits of specific codons can be examined for loss-of-function orgain-of-function based on specific polymorphisms in one or more codonsof the gene. These libraries can be a powerful tool for determiningwhich genetic changes are silent or causal of a specific phenotype in acell or cell population. The edits of the codons may berandomly-generated or may be rationally-designed based on knownpolymorphisms and/or mutations that have been identified in the gene tobe analyzed. Moreover, using these techniques on two or more genes in asingle in a pathway in a cell may determine potential protein:proteininteractions or redundancies in cell functions or pathways.

For example, alanine scanning can be used to determine the contributionof a specific residue to the stability or function of given protein.See, e.g., Lefévre, et al., Nucleic Acids Research, 25(2):447-448(1997). Alanine is often used in this codon scanning technique becauseof its non-bulky, chemically inert, methyl functional group that canmimic the secondary structure preferences that many of the other aminoacids possess. Codon scanning can also be used to determine whether theside chain of a specific residue plays a significant role in cellfunction and/or activity. Sometimes other amino acids such as valine orleucine can be used in the creation of codon scanning cell libraries ifconservation of the size of mutated residues is needed.

In other specific aspects, cell libraries can be created using theautomated editing methods and multi-module cell editing instruments todetermine the active site of a protein such as an enzyme or hormone, andto elucidate the mechanism of action of one or more of these proteins ina cell library. Site-directed mutagenesis associated with molecularmodeling studies can be used to discover the active site structure of anenzyme and consequently its mechanism of action. Analysis of these celllibraries can provide an understanding of the role exerted by specificamino acid residues at the active sites of proteins, in the contactsbetween subunits of protein complexes on intracellular trafficking andprotein stability/half-life in various genetic backgrounds.

Saturation Mutagenesis

In some aspects, the cell libraries created using the automated editingmethods and multi-module cell editing instruments of the disclosure maybe saturation mutagenesis libraries, in which a single codon or set ofcodons is randomized to produce all possible amino acids at the positionof a particular gene or genes of interest. These cell libraries can beparticularly useful to generate variants, e.g., for directed evolution.See, e.g., Chica, et al., Current Opinion in Biotechnology, 16(4):378-384 (2005); and Shivange, Current Opinion in Chemical Biology,13(1): 19-25 (2009).

In some aspects, edits comprising different degenerate codons can beused to encode sets of amino acids in the individual cells in thelibraries. Because some amino acids are encoded by more codons thanothers, the exact ratio of amino acids cannot be equal. In certainaspects, more restricted degenerate codons are used. ‘NNK’ and ‘NNS’have the benefit of encoding all 20 amino acids, but still encode a stopcodon 3% of the time. Alternative codons such as ‘NDT’, ‘DBK’ avoid stopcodons entirely, and encode a minimal set of amino acids that stillencompass all the main biophysical types (anionic, cationic, aliphatichydrophobic, aromatic hydrophobic, hydrophilic, small).

Promoter Swaps and Ladders

One mechanism for analyzing and/or optimizing expression of one or moregenes of interest is through the creation of a “promoter swap” celllibrary, in which the cells comprise genetic edits that have specificpromoters linked to one or more genes of interest. Accordingly, celllibraries created using the automated methods and multi-module cellediting instruments may be promoter swap cell libraries, which is used,e.g., to increase or decrease expression of a gene of interest tooptimize a metabolic or genetic pathway. In some aspects, the promoterswap cell library is used to identify an increase or reduction in theexpression of a gene that affects cell vitality or viability, e.g., agene encoding a protein that impacts on the growth rate or overallhealth of the cells. In some aspects, the promoter swap cell library canbe used to create cells having dependencies and logic between thepromoters to create synthetic gene networks. In some aspects, thepromoter swaps can be used to control cell to cell communication betweencells of both homogeneous and heterogeneous (complex tissues)populations in nature.

The cell libraries may utilize any given number of promoters that havebeen grouped together based upon exhibition of a range of expressionstrengths and any given number of target genes. The ladder of promotersequences vary expression of at least one locus under at least onecondition. This ladder is then systematically applied to a group ofgenes on the shuttle vector using the automated editing methods andmulti-module cell editing instruments of the disclosure.

In specific aspects, the cell library formed using the automated editingprocesses, modules and multi-module instruments of the disclosureinclude individual cells that are representative of a given promoteroperably linked to one or more target genes of interest in an otherwiseidentical genetic background.

In specific aspects, the promoter swap cell library is produced byediting a set of target genes on a shuttle vector to be operably linkedto a pre-selected set of promoters that act as a “promoter ladder” forexpression of the genes of interest. For example, the shuttle vectorsare edited so that one or more individual genes of interest are editedto be operably linked with the different promoters in the promoterladder. When an endogenous promoter does not exist, its sequence isunknown, or it has been previously changed in some manner, theindividual promoters of the promoter ladder can be inserted in front ofthe genes of interest. These produced cell libraries have individualcells with an individual promoter of the ladder operably linked to oneor more target genes in a shuttle vector in an otherwise identicalgenetic context. The promoters are generally selected to result invariable expression across different loci, and may include induciblepromoters, constitutive promoters, or both.

The set of target genes edited using the promoter ladder can include allor most open reading frames (ORFs) in a shuttle vector, or a selectedsubset of the shuttle vector. In some aspects, the target genes caninclude coding regions for various isoforms of the genes, and the celllibraries can be designed to express one or more specific isoforms,e.g., for transcriptome analysis using various promoters.

The set of target genes can also be genes known or suspected to beinvolved in a particular cellular pathway, e.g. a regulatory pathway orsignaling pathway. The set of target genes can be ORFs related tofunction, by relation to previously-demonstrated beneficial edits(previous promoter swaps or previous SNP swaps), by algorithmicselection based on epistatic interactions between previously generatededits, other selection criteria based on hypotheses regarding beneficialORF to target. In specific embodiments, the target genes can comprisenon-protein coding genes, including non-coding RNAs.

Editing of other functional genetic elements, including insulatorelements and other genomic organization elements, can also be used tosystematically vary the expression level of a set of target genes, andcan be introduced using the automated methods and automated multi-modulecell editing instruments of the disclosure. In one aspect, a populationof cells comprising shuttle vectors is edited using a ladder of enhancersequences, either alone or in combination with selected promoters or apromoter ladder, to create a cell library having various edits in theseenhancer elements. In another aspect, a population of cells is editedusing a ladder of ribosome binding sequences, either alone or incombination with selected promoters or a promoter ladder, to create acell library having various edits in these ribosome binding sequences.

In another aspect, a population of cells is edited to allow theattachment of various mRNA and/or protein stabilizing or destabilizingsequences to the 5′ or 3′ end, or at any other location, of a transcriptor protein.

When recursive editing is used, the editing in the individual cells inthe edited cell library can incorporate the inclusion of “landing pads”in an ectopic site in the shuttle vector to optimize expression,stability and/or control.

In some embodiments, each library produced having shuttle vectorscomprising one or more edits (either introducing or removing) iscultured and analyzed under one or more criteria (e.g., production of achemical or product of interest). The cells possessing the specificcriteria are then associated, or correlated, with one or more particularedits in the cell. In this manner, the effect of a given edit on anynumber of genetic or phenotypic traits of interest can be determined.The identification of multiple edits associated with particular criteriaor enhanced functionality/robustness may lead to cells with highlydesirable characteristics.

Knock-Out or Knock-in Libraries

In certain aspects, the present disclosure provides automated editingmethods, and automated modules, instruments and systems for creating alibrary of cells comprising shuttle vectors having “knock-out” (KO) or“knock-in” (KI) edits of various genes of interest. Thus, the disclosureis intended to cover edited cell libraries created by the automatedediting methods and automated multi-module cell editing instruments ofthe disclosure that have one or more mutations that remove or reduce theexpression of selected genes of interest to interrogate the effect ofthese edits on gene function in individual cells within the celllibrary.

The cell libraries comprising the shuttle vectors can be created usingtargeted gene KO (e.g., via insertion/deletion) or KOs (e.g., viahomologous directed repair). For example, double strand breaks are oftenrepaired via the non-homologous end joining DNA repair pathway. Therepair is known to be error prone, and thus insertions and deletions maybe introduced that can disrupt gene function. Preferably the edits arerationally designed to specifically affect the genes of interest, andindividual cells can be created having a KI or KI of one or more locusof interest. Cells having a KO or KI of two or more loci of interest canbe created using automated recursive editing of the disclosure.

In specific aspects, the KO or KI cell libraries are created usingsimultaneous multiplexed editing of shuttle vectors within a cellpopulation, and multiple shuttle vectors within a cell population areedited in a single round of editing, i.e., multiple changes within theshuttle vectors of the cells of the cell library are in a singleautomated operation. In other specific aspects, the cell libraries arecreated using recursive editing of shuttle vectors within a cellpopulation, and results in the amalgamation of multiple edits of two ormore sites in the shuttle vectors into single cells.

SNP or Short Tandem Repeat Swaps

In one aspect, cell libraries are created using the automated editingmethods and automated multi-module cell editing instruments of thedisclosure by systematically introducing or substituting singlenucleotide polymorphisms (“SNPs”) into the shuttle vectors of theindividual cells to create a “SNP swap” cell library. In someembodiments, the SNP swapping methods include both the addition ofbeneficial SNPs and removing detrimental and/or neutral SNPs. The SNPswaps may target coding sequences, non-coding sequences, or both.

In another aspect, a cell library is created using the automated editingmethods and modules, instruments, and systems by systematicallyintroducing or substituting short tandem repeats (“STR”) into theshuttle vectors of the individual cells to create an “STR swap” celllibrary. In some embodiments, the STR swapping methods of the presentdisclosure include both the addition of beneficial STRs and removingdetrimental and/or neutral STRs. The STR swaps may target codingsequences, non-coding sequences, or both.

In some embodiments, the SNP and/or STR swapping used to create the celllibrary is multiplexed, and multiple shuttle vectors in the cells withina cell population are edited in a single round of editing, i.e.,multiple changes within the shuttle vectors in the cells of the celllibrary are introduced in a single automated operation. In otherembodiments, the SNP and/or STR swapping used to create the cell libraryis recursive, and results in the amalgamation of multiple beneficialsequences and/or the removal of detrimental sequences into the shuttlevectors. Multiple changes can be either a specific set of definedchanges or a partly-randomized, combinatorial library of mutations.Removal of detrimental mutations and consolidation of beneficialmutations can provide immediate improvements in various cellularprocesses. Removal of genetic burden or consolidation of beneficialchanges into a cell with no genetic burden also provides a new, robuststarting point for additional random mutagenesis that may enable furtherimprovements.

SNP swapping overcomes fundamental limitations of random mutagenesisapproaches as it is not a random approach, but rather the systematicintroduction or removal of individual mutations across cells.

Splice Site Editing

RNA splicing is the process during which introns are excised and exonsare spliced together to create the mRNA that is translated into aprotein. The precise recognition of splicing signals by cellularmachinery is critical to this process. Accordingly, in some aspects, apopulation of cells comprising one or more shuttle vectors is editedusing systematic editing of known and/or predicted splice donor and/oracceptor sites in various loci to create a library of splice sitevariants of various genes. Such editing can help to elucidate thebiological relevance of various isoforms of genes in a cellular context.Sequences for rational design of splicing sites of various codingregions, including actual or predicted mutations associated with variousmammalian disorders, can be predicted using analysis techniques such asthose found in Nalla and Rogan, Hum. Mutat., 25:334-342 (2005); Divina,et al., Eur. J. Hum. Genet., 17:759-765 (2009); Desmet, et el., NucleicAcids Res, 37:e67 (2009); Faber, et al., BMC Bioinformatics, 12(suppl4):S2 (2011).

Start/Stop Codon Exchanges and Incorporation of Nucleic Acid Analogs

In some aspects, there is provided creation of cell libraries comprisingshuttle vectors using the automated editing methods and modules,instruments and systems of the disclosure, where the libraries arecreated by swapping start and stop codon variants. For example, typicalstart codons used by eukaryotes are ATG (AUG) most frequently, followedby GTG (GUG) and TTG (UUG). The cell library may include individualcells having substitutions for the native start codons for one or moregenes of interest expressed from the shuttle vector. In some aspects,there is provided automated creation of a cell library by replacing ATGstart codons with TTG in front of selected genes of interest. In otheraspects, there is provided automated creation of a cell library byreplacing ATG start codons with GTG. In other aspects, there is providedautomated creation of a cell library by replacing GTG start codons withATG. In other aspects, there is provided automated creation of a celllibrary by replacing GTG start codons with TTG. In other aspects, thereis provided automated creation of a cell library by replacing TTG startcodons with ATG. In other aspects, there is provided automated creationof a cell library by replacing TTG start codons with GTG.

In other examples, typical stop codons for S. cerevisiae and mammals areTAA (UAA) and TGA (UGA), respectively. The typical stop codon formonocotyledonous plants is TGA (UGA), whereas insects commonly use TAA(UAA) as the stop codon (Dalphin, et al., Nucl. Acids Res., 24: 216-218(1996)). The cell library may include individual cells havingsubstitutions for the native stop codons for one or more genes ofinterest. In some aspects, there is provided automated creation of acell library by replacing TAA stop codons with TAG. In other aspects,there is provided a cell library by replacing TAA stop codons with TGA.In other aspects, there is provided a cell library by replacing TGA stopcodons with TAA. In other aspects, there is provided a cell library byreplacing TGA stop codons with TAG. In other aspects, there is provideda cell library by replacing TAG stop codons with TAA. In other aspects,there is provided automated creation of a cell library by replacing TAGstop codons with TGA.

Terminator Swaps and Ladders

One mechanism for identifying optimum termination of a pre-spliced mRNAof one or more genes of interest is through the creation of a“terminator swap” cell library, in which the cells comprise shuttlevectors that comprise genetic edits that have specific terminatorsequences linked to one or more genes of interest. Accordingly, the celllibraries created using the automated methods, modules, instruments andsystems of the disclosure may be terminator swap cell libraries, whichcan be used, e.g., to affect mRNA stability by releasing transcriptsfrom sites of synthesis. In other embodiments, the terminator swap celllibrary can be used to identify an increase or reduction in theefficiency of transcriptional termination and thus accumulation ofunspliced pre-mRNA (e.g., West and Proudfoot, Mol Cell., 33(3-9);354-364 (2009)) and/or 3′ end processing (e.g., West, et al., Mol Cell.29(5):600-10 (2008)). In the case where a gene is linked to multipletermination sites, the edits may edit a combination of edits to multipleterminators that are associated with a gene. Additional amino acids mayalso be added to the ends of proteins to determine the effect on theprotein length on terminators.

The cell libraries comprising shuttle vectors utilize any given numberof edits of terminators that have been selected for the terminatorladder based upon exhibition of a range of activity and any given numberof target genes. The ladder of terminator sequences vary expression ofat least one locus under at least one condition. This ladder is thensystematically applied to a group of genes in the organism using theautomated editing methods, modules, instruments and systems of thedisclosure.

In some aspects, there is provided creation of cell libraries using theautomated editing methods, modules, instruments and systems ofdisclosure, where the libraries are created to edit terminator signalsin one or more regions in the shuttle vector(s) in the individual cellsof the library. Transcriptional termination in eukaryotes operatesthrough terminator signals that are recognized by protein factorsassociated with the RNA polymerase II. For example, the cell library maycontain individual eukaryotic cells with edits in genes encodingpolyadenylation specificity factor (CPSF) and cleavage stimulationfactor (CstF) and/or genes encoding proteins recruited by CPSF and CstFfactors to termination sites.

In certain aspects, the present disclosure provides methods of selectingtermination sequences (“terminators”) with optimal properties. Forexample, in some embodiments, provided are methods for introducingand/or editing one or more terminators and/or generating variants of oneor more terminators within a heterologous editing cell, which exhibit arange of activity.

In specific aspects, the terminator swap cell library is produced byediting a set of target genes in a shuttle vector to be operably linkedto a pre-selected set of terminators that act as a “terminator ladder”for expression of the genes of interest. For example, the cells areedited so that the endogenous promoter is operably linked to theindividual genes of interest, which are then edited with the differentpromoters in the promoter ladder. When the endogenous promoter does notexist, its sequence is unknown, or it has been previously changed insome manner, the individual promoters of the promoter ladder can beinserted in front of the genes of interest. These cell libraries haveindividual cells with one or more shuttle vectors comprising with anindividual promoter of the ladder operably linked to one or more targetgenes in an otherwise identical genetic context. The terminator ladderin question is then associated with a given gene of interest. Theterminator ladder can be used to more generally affect termination ofall or most ORFs in a shuttle vector, or a selected subset of theshuttle vector. The set of target genes can also be genes known orsuspected to be involved in a particular cellular pathway, e.g. aregulatory pathway or signaling pathway. The set of target genes can beORFs related to function, by relation to previously demonstratedbeneficial edits (previous promoter swaps or previous SNP swaps), byalgorithmic selection based on epistatic interactions between previouslygenerated edits, other selection criteria based on hypotheses regardingbeneficial ORF to target, or through random selection. In specificembodiments, the target genes can comprise non-protein coding genes,including non-coding RNAs.

Automated Cell Editing Instruments and Modules to Perform NucleicAcid-Guided Nuclease Editing in Cells Automated Cell Editing Instruments

FIG. 2A depicts an exemplary automated multi-module cell processinginstrument 200 to, e.g., perform one of the exemplary workflows fortargeted gene editing of live cells. The instrument 200, for example,may be and preferably is designed as a stand-alone desktop instrumentfor use within a laboratory environment. The instrument 200 mayincorporate a mixture of reusable and disposable components forperforming the various integrated processes in conducting automatedgenome cleavage and/or editing in cells without human intervention.Illustrated is a gantry 202, providing an automated mechanical motionsystem (actuator) (not shown) that supplies XYZ axis motion control to,e.g., an automated (i.e., robotic) liquid handling system 258 including,e.g., an air displacement pipettor 232 which allows for cell processingamong multiple modules without human intervention. In some automatedmulti-module cell processing instruments, the air displacement pipettor232 is moved by gantry 202 and the various modules and reagentcartridges remain stationary; however, in other embodiments, the liquidhandling system 258 may stay stationary while the various modules andreagent cartridges are moved. Also included in the automatedmulti-module cell processing instrument 200 are reagent cartridges 210comprising reservoirs 212 and transformation module 230 (e.g., aflow-through electroporation device as described in detail in relationto FIGS. 5B-5F), as well as wash reservoirs 206, cell input reservoir251 and cell output reservoir 253. The wash reservoirs 206 may beconfigured to accommodate large tubes, for example, wash solutions, orsolutions that are used often throughout an iterative process. Althoughtwo of the reagent cartridges 210 comprise a wash reservoir 206 in FIG.2A, the wash reservoirs instead could be included in a wash cartridgewhere the reagent and wash cartridges are separate cartridges. In such acase, the reagent cartridge 210 and wash cartridge 204 may be identicalexcept for the consumables (reagents or other components containedwithin the various inserts) inserted therein.

In some implementations, the reagent cartridges 210 are disposable kitscomprising reagents and cells for use in the automated multi-module cellprocessing/editing instrument 200. For example, a user may open andposition each of the reagent cartridges 210 comprising various desiredinserts and reagents within the chassis of the automated multi-modulecell editing instrument 200 prior to activating cell processing.Further, each of the reagent cartridges 210 may be inserted intoreceptacles in the chassis having different temperature zonesappropriate for the reagents contained therein.

Also illustrated in FIG. 2A is the robotic liquid handling system 258including the gantry 202 and air displacement pipettor 232. In someexamples, the robotic handling system 258 may include an automatedliquid handling system such as those manufactured by Tecan Group Ltd. ofMannedorf, Switzerland, Hamilton Company of Reno, Nev. (see, e.g.,WO2018015544A1), or Beckman Coulter, Inc. of Fort Collins, Colo. (see,e.g., US20160018427A1). Pipette tips may be provided in a pipettetransfer tip supply (not shown) for use with the air displacementpipettor 232.

Inserts or components of the reagent cartridges 210, in someimplementations, are marked with machine-readable indicia (not shown),such as bar codes, for recognition by the robotic handling system 258.For example, the robotic liquid handling system 258 may scan one or moreinserts within each of the reagent cartridges 210 to confirm contents.In other implementations, machine-readable indicia may be marked uponeach reagent cartridge 210, and a processing system (not shown, but seeelement 237 of FIG. 2B) of the automated multi-module cell editinginstrument 200 may identify a stored materials map based upon themachine-readable indicia. In the embodiment illustrated in FIG. 2A, acell growth module comprises a cell growth vial 218 (described ingreater detail below in relation to FIGS. 3A-3D). Additionally seen isthe TFF module 222 (described above in detail in relation to FIGS.4A-4E). Also illustrated as part of the automated multi-module cellprocessing instrument 200 of FIG. 2A is a singulation module 240 (e.g.,a solid wall isolation, incubation and normalization device (SWIINdevice) is shown here) described herein in relation to FIGS. 6C-6F,served by, e.g., robotic liquid handing system 258 and air displacementpipettor 232. Additionally seen is a selection module 220. Also note theplacement of three heatsinks 255.

FIG. 2B is a simplified representation of the contents of the exemplarymulti-module cell processing instrument 200 depicted in FIG. 2A.Cartridge-based source materials (such as in reagent cartridges 210),for example, may be positioned in designated areas on a deck of theinstrument 200 for access by an air displacement pipettor 232. The deckof the multi-module cell processing instrument 200 may include aprotection sink such that contaminants spilling, dripping, oroverflowing from any of the modules of the instrument 200 are containedwithin a lip of the protection sink. Also seen are reagent cartridges210, which are shown disposed with thermal assemblies 211 which cancreate temperature zones appropriate for different regions. Note thatone of the reagent cartridges also comprises a flow-throughelectroporation device 230 (FTEP), served by FTEP interface (e.g.,manifold arm) and actuator 231. Also seen is TFF module 222 withadjacent thermal assembly 225, where the TFF module is served by TFFinterface (e.g., manifold arm) and actuator 233. Thermal assemblies 225,235, and 245 encompass thermal electric devices such as Peltier devices,as well as heatsinks, fans and coolers. The rotating growth vial 218 iswithin a growth module 234, where the growth module is served by twothermal assemblies 235. Selection module is seen at 220. Also seen isthe SWIIN module 240, comprising a SWIIN cartridge 241, where the SWIINmodule also comprises a thermal assembly 245, illumination 243 (in thisembodiment, backlighting), evaporation and condensation control 249, andwhere the SWIIN module is served by SWIIN interface (e.g., manifold arm)and actuator 247. Also seen in this view is touch screen display 201,display actuator 203, illumination 205 (one on either side ofmulti-module cell processing instrument 200), and cameras 239 (oneillumination device on either side of multi-module cell processinginstrument 200). Finally, element 237 comprises electronics, such ascircuit control boards, high-voltage amplifiers, power supplies, andpower entry; as well as pneumatics, such as pumps, valves and sensors.

FIG. 2C illustrates a front perspective view of multi-module cellprocessing instrument 200 for use in as a desktop version of theautomated multi-module cell editing instrument 200. For example, achassis 290 may have a width of about 24-48 inches, a height of about24-48 inches and a depth of about 24-48 inches. Chassis 290 may be andpreferably is designed to hold all modules and disposable supplies usedin automated cell processing and to perform all processes requiredwithout human intervention; that is, chassis 290 is configured toprovide an integrated, stand-alone automated multi-module cellprocessing instrument. As illustrated in FIG. 2C, chassis 290 includestouch screen display 201, cooling grate 264, which allows for air flowvia an internal fan (not shown). The touch screen display providesinformation to a user regarding the processing status of the automatedmulti-module cell editing instrument 200 and accepts inputs from theuser for conducting the cell processing. In this embodiment, the chassis290 is lifted by adjustable feet 270 a, 270 b, 270 c and 270 d (feet 270a-270 c are shown in this FIG. 2C). Adjustable feet 270 a-270 d, forexample, allow for additional air flow beneath the chassis 290.

Inside the chassis 290, in some implementations, will be most or all ofthe components described in relation to FIGS. 2A and 2B, including therobotic liquid handling system disposed along a gantry, reagentcartridges 210 including a flow-through electroporation device, arotating growth vial 218 in a cell growth module 234, a tangential flowfiltration module 222, a SWIIN module 240 as well as interfaces andactuators for the various modules. In addition, chassis 290 housescontrol circuitry, liquid handling tubes, air pump controls, valves,sensors, thermal assemblies (e.g., heating and cooling units) and othercontrol mechanisms. For examples of multi-module cell editinginstruments, see U.S. Pat. No. 10,253,316, issued 9 Apr. 2019; U.S. Pat.No. 10,329,559, issued 25 Jun. 2019; U.S. Pat. No. 10,323,242, issued 18Jun. 2019; U.S. Pat. No. 10,421,959, issued 24 Sep. 2019; U.S. Pat. No.10,465,185, issued 5 Nov. 2019; U.S. Pat. No. 10,519,437, issued 31 Dec.2019 and U.S. Ser. No. 16/412,195, filed 14 May 2019; Ser. No.16/680,643, filed 12 Nov. 2019; and Ser. No. 16/750,369, filed 23 Jan.2020, all of which are herein incorporated by reference in theirentirety.

The Rotating Cell Growth Module

FIG. 3A shows one embodiment of a rotating growth vial 300 for use withthe cell growth device and in the automated multi-module cell processinginstruments described herein. The rotating growth vial 300 is anoptically-transparent container having an open end 304 for receivingliquid media and cells, a central vial region 306 that defines theprimary container for growing cells, a tapered-to-constricted region 318defining at least one light path 310, a closed end 316, and a driveengagement mechanism 312. The rotating growth vial 300 has a centrallongitudinal axis 320 around which the vial rotates, and the light path310 is generally perpendicular to the longitudinal axis of the vial. Thefirst light path 310 is positioned in the lower constricted portion ofthe tapered-to-constricted region 318. Optionally, some embodiments ofthe rotating growth vial 300 have a second light path 308 in the taperedregion of the tapered-to-constricted region 318. Both light paths inthis embodiment are positioned in a region of the rotating growth vialthat is constantly filled with the cell culture (cells+growth media) andare not affected by the rotational speed of the growth vial. The firstlight path 310 is shorter than the second light path 308 allowing forsensitive measurement of OD values when the OD values of the cellculture in the vial are at a high level (e.g., later in the cell growthprocess), whereas the second light path 308 allows for sensitivemeasurement of OD values when the OD values of the cell culture in thevial are at a lower level (e.g., earlier in the cell growth process).

The drive engagement mechanism 312 engages with a motor (not shown) torotate the vial. In some embodiments, the motor drives the driveengagement mechanism 312 such that the rotating growth vial 300 isrotated in one direction only, and in other embodiments, the rotatinggrowth vial 300 is rotated in a first direction for a first amount oftime or periodicity, rotated in a second direction (i.e., the oppositedirection) for a second amount of time or periodicity, and this processmay be repeated so that the rotating growth vial 300 (and the cellculture contents) are subjected to an oscillating motion. Further, thechoice of whether the culture is subjected to oscillation and theperiodicity therefor may be selected by the user. The first amount oftime and the second amount of time may be the same or may be different.The amount of time may be 1, 2, 3, 4, 5, or more seconds, or may be 1,2, 3, 4 or more minutes. In another embodiment, in an early stage ofcell growth the rotating growth vial 400 may be oscillated at a firstperiodicity (e.g., every 60 seconds), and then a later stage of cellgrowth the rotating growth vial 300 may be oscillated at a secondperiodicity (e.g., every one second) different from the firstperiodicity.

The rotating growth vial 300 may be reusable or, preferably, therotating growth vial is consumable. In some embodiments, the rotatinggrowth vial is consumable and is presented to the user pre-filled withgrowth medium, where the vial is hermetically sealed at the open end 304with a foil seal. A medium-filled rotating growth vial packaged in sucha manner may be part of a kit for use with a stand-alone cell growthdevice or with a cell growth module that is part of an automatedmulti-module cell processing system. To introduce cells into the vial, auser need only pipette up a desired volume of cells and use the pipettetip to punch through the foil seal of the vial. Open end 304 mayoptionally include an extended lip 402 to overlap and engage with thecell growth device. In automated systems, the rotating growth vial 400may be tagged with a barcode or other identifying means that can be readby a scanner or camera (not shown) that is part of the automated system.

The volume of the rotating growth vial 300 and the volume of the cellculture (including growth medium) may vary greatly, but the volume ofthe rotating growth vial 300 must be large enough to generate aspecified total number of cells. In practice, the volume of the rotatinggrowth vial 300 may range from 1-250 mL, 2-100 mL, from 5-80 mL, 10-50mL, or from 12-35 mL. Likewise, the volume of the cell culture(cells+growth media) should be appropriate to allow proper aeration andmixing in the rotating growth vial 300. Proper aeration promotes uniformcellular respiration within the growth media. Thus, the volume of thecell culture should be approximately 5-85% of the volume of the growthvial or from 20-60% of the volume of the growth vial. For example, for a30 mL growth vial, the volume of the cell culture would be from about1.5 mL to about 26 mL, or from 6 mL to about 18 mL.

The rotating growth vial 300 preferably is fabricated from abio-compatible optically transparent material—or at least the portion ofthe vial comprising the light path(s) is transparent. Additionally,material from which the rotating growth vial is fabricated should beable to be cooled to about 4° C. or lower and heated to about 55° C. orhigher to accommodate both temperature-based cell assays and long-termstorage at low temperatures. Further, the material that is used tofabricate the vial must be able to withstand temperatures up to 55° C.without deformation while spinning. Suitable materials include cyclicolefin copolymer (COC), glass, polyvinyl chloride, polyethylene,polyamide, polypropylene, polycarbonate, poly(methyl methacrylate(PMMA), polysulfone, polyurethane, and co-polymers of these and otherpolymers. Preferred materials include polypropylene, polycarbonate, orpolystyrene. In some embodiments, the rotating growth vial isinexpensively fabricated by, e.g., injection molding or extrusion.

FIG. 3B is a perspective view of one embodiment of a cell growth device330. FIG. 3C depicts a cut-away view of the cell growth device 330 fromFIG. 3B. In both figures, the rotating growth vial 300 is seenpositioned inside a main housing 336 with the extended lip 302 of therotating growth vial 300 extending above the main housing 336.Additionally, end housings 352, a lower housing 332 and flanges 334 areindicated in both figures. Flanges 334 are used to attach the cellgrowth device 330 to heating/cooling means or other structure (notshown). FIG. 3C depicts additional detail. In FIG. 3C, upper bearing 342and lower bearing 340 are shown positioned within main housing 336.Upper bearing 342 and lower bearing 340 support the vertical load ofrotating growth vial 300. Lower housing 332 contains the drive motor338. The cell growth device 330 of FIG. 3C comprises two light paths: aprimary light path 344, and a secondary light path 350. Light path 344corresponds to light path 310 positioned in the constricted portion ofthe tapered-to-constricted portion of the rotating growth vial 300, andlight path 350 corresponds to light path 308 in the tapered portion ofthe tapered-to-constricted portion of the rotating growth via 316. Lightpaths 310 and 308 are not shown in FIG. 3C but may be seen in FIG. 3A.In addition to light paths 344 and 340, there is an emission board 348to illuminate the light path(s), and detector board 346 to detect thelight after the light travels through the cell culture liquid in therotating growth vial 300.

The motor 338 engages with drive mechanism 312 and is used to rotate therotating growth vial 300. In some embodiments, motor 338 is a brushlessDC type drive motor with built-in drive controls that can be set to holda constant revolution per minute (RPM) between 0 and about 3000 RPM.Alternatively, other motor types such as a stepper, servo, brushed DC,and the like can be used. Optionally, the motor 338 may also havedirection control to allow reversing of the rotational direction, and atachometer to sense and report actual RPM. The motor is controlled by aprocessor (not shown) according to, e.g., standard protocols programmedinto the processor and/or user input, and the motor may be configured tovary RPM to cause axial precession of the cell culture thereby enhancingmixing, e.g., to prevent cell aggregation, increase aeration, andoptimize cellular respiration.

Main housing 336, end housings 352 and lower housing 332 of the cellgrowth device 330 may be fabricated from any suitable, robust materialincluding aluminum, stainless steel, and other thermally conductivematerials, including plastics. These structures or portions thereof canbe created through various techniques, e.g., metal fabrication,injection molding, creation of structural layers that are fused, etc.Whereas the rotating growth vial 300 is envisioned in some embodimentsto be reusable, but preferably is consumable, the other components ofthe cell growth device 330 are preferably reusable and function as astand-alone benchtop device or as a module in a multi-module cellprocessing system.

The processor (not shown) of the cell growth device 330 may beprogrammed with information to be used as a “blank” or control for thegrowing cell culture. A “blank” or control is a vessel containing cellgrowth medium only, which yields 100% transmittance and 0 OD, while thecell sample will deflect light rays and will have a lower percenttransmittance and higher OD. As the cells grow in the media and becomedenser, transmittance will decrease and OD will increase. The processor(not shown) of the cell growth device 330—may be programmed to usewavelength values for blanks commensurate with the growth mediatypically used in cell culture (whether, e.g., mammalian cells,bacterial cells, animal cells, yeast cells, etc.). Alternatively, asecond spectrophotometer and vessel may be included in the cell growthdevice 330, where the second spectrophotometer is used to read a blankat designated intervals.

FIG. 3D illustrates a cell growth device 330 as part of an assemblycomprising the cell growth device 330 of FIG. 3B coupled to light source390, detector 392, and thermal components 394. The rotating growth vial300 is inserted into the cell growth device. Components of the lightsource 390 and detector 392 (e.g., such as a photodiode with gaincontrol to cover 5-log) are coupled to the main housing of the cellgrowth device. The lower housing 332 that houses the motor that rotatesthe rotating growth vial 300 is illustrated, as is one of the flanges334 that secures the cell growth device 330 to the assembly. Also, thethermal components 394 illustrated are a Peltier device orthermoelectric cooler. In this embodiment, thermal control isaccomplished by attachment and electrical integration of the cell growthdevice 330 to the thermal components 394 via the flange 334 on the baseof the lower housing 332. Thermoelectric coolers are capable of“pumping” heat to either side of a junction, either cooling a surface orheating a surface depending on the direction of current flow. In oneembodiment, a thermistor is used to measure the temperature of the mainhousing and then, through a standard electronicproportional-integral-derivative (PID) controller loop, the rotatinggrowth vial 300 is controlled to approximately +/−0.5° C.

In use, cells are inoculated (cells can be pipetted, e.g., from anautomated liquid handling system or by a user) into pre-filled growthmedia of a rotating growth vial 300 by piercing though the foil seal orfilm. The programmed software of the cell growth device 330 sets thecontrol temperature for growth, typically 30° C., then slowly starts therotation of the rotating growth vial 300. The cell/growth media mixtureslowly moves vertically up the wall due to centrifugal force allowingthe rotating growth vial 300 to expose a large surface area of themixture to a normal oxygen environment. The growth monitoring systemtakes either continuous readings of the OD or OD measurements at pre-setor pre-programmed time intervals. These measurements are stored ininternal memory and if requested the software plots the measurementsversus time to display a growth curve. If enhanced mixing is required,e.g., to optimize growth conditions, the speed of the vial rotation canbe varied to cause an axial precession of the liquid, and/or a completedirectional change can be performed at programmed intervals. The growthmonitoring can be programmed to automatically terminate the growth stageat a pre-determined OD, and then quickly cool the mixture to a lowertemperature to inhibit further growth.

One application for the cell growth device 330 is to constantly measurethe optical density of a growing cell culture. One advantage of thedescribed cell growth device is that optical density can be measuredcontinuously (kinetic monitoring) or at specific time intervals; e.g.,every 5, 10, 15, 20, 30 45, or 60 seconds, or every 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 minutes. While the cell growth device 330 has been describedin the context of measuring the optical density (OD) of a growing cellculture, it should, however, be understood by a skilled artisan giventhe teachings of the present specification that other cell growthparameters can be measured in addition to or instead of cell culture OD.As with optional measure of cell growth in relation to the solid walldevice or module described supra, spectroscopy using visible, UV, ornear infrared (NIR) light allows monitoring the concentration ofnutrients and/or wastes in the cell culture and other spectroscopicmeasurements may be made; that is, other spectral properties can bemeasured via, e.g., dielectric impedance spectroscopy, visiblefluorescence, fluorescence polarization, or luminescence. Additionally,the cell growth device 430 may include additional sensors for measuring,e.g., dissolved oxygen, carbon dioxide, pH, conductivity, and the like.For additional details regarding rotating growth vials and cell growthdevices see U.S. Pat. No. 10,435,662, issued 8 Oct. 2019; U.S. Pat. No.10,443,031, issued 15 Oct. 2019; and U.S. Ser. No. 16/552,981, filed 7Aug. 2019.

The Cell Concentration Module

As described above in relation to the rotating growth vial and cellgrowth module, in order to obtain an adequate number of cells fortransformation or transfection, cells typically are grown to a specificoptical density in medium appropriate for the growth of the cells ofinterest; however, for effective transformation or transfection, it isdesirable to decrease the volume of the cells as well as render thecells competent via buffer or medium exchange. Thus, one sub-componentor module that is desired in cell processing systems for the processeslisted above is a module or component that can grow, perform bufferexchange, and/or concentrate cells and render them competent so thatthey may be transformed or transfected with the nucleic acids needed forengineering or editing the cell's genome.

FIG. 4A shows a retentate member 422 (top), permeate member 420 (middle)and a tangential flow assembly 410 (bottom) comprising the retentatemember 422, membrane 424 (not seen in FIG. 4A), and permeate member 420(also not seen). In FIG. 4A, retentate member 422 comprises a tangentialflow channel 402, which has a serpentine configuration that initiates atone lower corner of retentate member 422—specifically at retentate port428—traverses across and up then down and across retentate member 422,ending in the other lower corner of retentate member 422 at a secondretentate port 428. Also seen on retentate member 422 are energydirectors 491, which circumscribe the region where a membrane or filter(not seen in this FIG. 4A) is seated, as well as interdigitate betweenareas of channel 402. Energy directors 491 in this embodiment mate withand serve to facilitate ultrasonic welding or bonding of retentatemember 422 with permeate/filtrate member 420 via the energy directorcomponent 491 on permeate/filtrate member 420 (at right). Additionally,countersinks 423 can be seen, two on the bottom one at the top middle ofretentate member 422. Countersinks 423 are used to couple and tangentialflow assembly 410 to a reservoir assembly (not seen in this FIG. 4A butsee FIG. 4B).

Permeate/filtrate member 420 is seen in the middle of FIG. 4A andcomprises, in addition to energy director 491, through-holes forretentate ports 428 at each bottom corner (which mate with thethrough-holes for retentate ports 428 at the bottom corners of retentatemember 422), as well as a tangential flow channel 402 and twopermeate/filtrate ports 426 positioned at the top and center of permeatemember 420. The tangential flow channel 402 structure in this embodimenthas a serpentine configuration and an undulating geometry, althoughother geometries may be used. Permeate member 420 also comprisescountersinks 423, coincident with the countersinks 423 on retentatemember 420.

On the left of FIG. 4A is a tangential flow assembly 410 comprising theretentate member 422 and permeate member 420 seen in this FIG. 4A. Inthis view, retentate member 422 is “on top” of the view, a membrane (notseen in this view of the assembly) would be adjacent and under retentatemember 422 and permeate member 420 (also not seen in this view of theassembly) is adjacent to and beneath the membrane. Again countersinks423 are seen, where the countersinks in the retentate member 422 and thepermeate member 420 are coincident and configured to mate with threadsor mating elements for the countersinks disposed on a reservoir assembly(not seen in FIG. 4A but see FIG. 4B).

A membrane or filter is disposed between the retentate and permeatemembers, where fluids can flow through the membrane but cells cannot andare thus retained in the flow channel disposed in the retentate member.Filters or membranes appropriate for use in the TFF device/module arethose that are solvent resistant, are contamination free duringfiltration, and are able to retain the types and sizes of cells ofinterest. For example, in order to retain small cell types such asbacterial cells, pore sizes can be as low as 0.2 μm, however for othercell types, the pore sizes can be as high as 20 μm. Indeed, the poresizes useful in the TFF device/module include filters with sizes from0.20 μm, 0.21 μm, 0.22 μm, 0.23 μm, 0.24 μm, 0.25 μm, 0.26 μm, 0.27 μm,0.28 μm, 0.29 μm, 0.30 μm, 0.31 μm, 0.32 μm, 0.33 μm, 0.34 μm, 0.35 μm,0.36 μm, 0.37 μm, 0.38 μm, 0.39 μm, 0.40 μm, 0.41 μm, 0.42 μm, 0.43 μm,0.44 μm, 0.45 μm, 0.46 μm, 0.47 μm, 0.48 μm, 0.49 μm, 0.50 μm andlarger. The filters may be fabricated from any suitable non-reactivematerial including cellulose mixed ester (cellulose nitrate and acetate)(CME), polycarbonate (PC), polyvinylidene fluoride (PVDF),polyethersulfone (PES), polytetrafluoroethylene (PTFE), nylon, glassfiber, or metal substrates as in the case of laser or electrochemicaletching.

The length of the channel structure 402 may vary depending on the volumeof the cell culture to be grown and the optical density of the cellculture to be concentrated. The length of the channel structuretypically is from 60 mm to 300 mm, or from 70 mm to 200 mm, or from 80mm to 100 mm. The cross-section configuration of the flow channel 402may be round, elliptical, oval, square, rectangular, trapezoidal, orirregular. If square, rectangular, or another shape with generallystraight sides, the cross section may be from about 10 μm to 1000 μmwide, or from 200 μm to 800 μm wide, or from 300 μm to 700 μm wide, orfrom 400 μm to 600 μm wide; and from about 10 μm to 1000 μm high, orfrom 200 μm to 800 μm high, or from 300 μm to 700 μm high, or from 400μm to 600 μm high. If the cross section of the flow channel 102 isgenerally round, oval or elliptical, the radius of the channel may befrom about 50 μm to 1000 μm in hydraulic radius, or from 5 μm to 800 μmin hydraulic radius, or from 200 μm to 700 μm in hydraulic radius, orfrom 300 μm to 600 μm wide in hydraulic radius, or from about 200 to 500μm in hydraulic radius. Moreover, the volume of the channel in theretentate 422 and permeate 420 members may be different depending on thedepth of the channel in each member.

FIG. 4B shows front perspective (right) and rear perspective (left)views of a reservoir assembly 450 configured to be used with thetangential flow assembly 410 seen in FIG. 4A. Seen in the frontperspective view (e.g., “front” being the side of reservoir assembly 450that is coupled to the tangential flow assembly 410 seen in FIG. 4A) areretentate reservoirs 452 on either side of permeate reservoir 454. Alsoseen are permeate ports 426, retentate ports 428, and three threads ormating elements 425 for countersinks 423 (countersinks 423 not seen inthis FIG. 4B). Threads or mating elements 425 for countersinks 423 areconfigured to mate or couple the tangential flow assembly 410 (seen inFIG. 4A) to reservoir assembly 450. Alternatively or in addition,fasteners, sonic welding or heat stakes may be used to mate or couplethe tangential flow assembly 410 to reservoir assembly 450. In additionis seen gasket 445 covering the top of reservoir assembly 450. Gasket445 is described in detail in relation to FIG. 4E. At left in FIG. 4B isa rear perspective view of reservoir assembly 1250, where “rear” is theside of reservoir assembly 450 that is not coupled to the tangentialflow assembly. Seen are retentate reservoirs 452, permeate reservoir454, and gasket 445.

The TFF device may be fabricated from any robust material in whichchannels (and channel branches) may be milled including stainless steel,silicon, glass, aluminum, or plastics including cyclic-olefin copolymer(COC), cyclo-olefin polymer (COP), polystyrene, polyvinyl chloride,polyethylene, polyamide, polyethylene, polypropylene, acrylonitrilebutadiene, polycarbonate, polyetheretheketone (PEEK), poly(methylmethylacrylate) (PMMA), polysulfone, and polyurethane, and co-polymersof these and other polymers. If the TFF device/module is disposable,preferably it is made of plastic. In some embodiments, the material usedto fabricate the TFF device/module is thermally-conductive so that thecell culture may be heated or cooled to a desired temperature. Incertain embodiments, the TFF device is formed by precision mechanicalmachining, laser machining, electro discharge machining (for metaldevices); wet or dry etching (for silicon devices); dry or wet etching,powder or sandblasting, photostructuring (for glass devices); orthermoforming, injection molding, hot embossing, or laser machining (forplastic devices) using the materials mentioned above that are amenableto this mass production techniques.

FIG. 4C depicts a top-down view of the reservoir assemblies 450 shown inFIG. 4B. FIG. 4D depicts a cover 444 for reservoir assembly 450 shown inFIGS. 4B and 4E depicts a gasket 445 that in operation is disposed oncover 444 of reservoir assemblies 450 shown in FIG. 4B. FIG. 4C is atop-down view of reservoir assembly 450, showing the tops of the tworetentate reservoirs 452, one on either side of permeate reservoir 454.Also seen are grooves 432 that will mate with a pneumatic port (notshown), and fluid channels 434 that reside at the bottom of retentatereservoirs 452, which fluidically couple the retentate reservoirs 452with the retentate ports 428 (not shown), via the through-holes for theretentate ports in permeate member 420 and membrane 424 (also notshown). FIG. 4D depicts a cover 444 that is configured to be disposedupon the top of reservoir assembly 450. Cover 444 has round cut-outs atthe top of retentate reservoirs 452 and permeate/filtrate reservoir 454.Again at the bottom of retentate reservoirs 452 fluid channels 434 canbe seen, where fluid channels 434 fluidically couple retentatereservoirs 452 with the retentate ports 428 (not shown). Also shown arethree pneumatic ports 430 for each retentate reservoir 452 andpermeate/filtrate reservoir 454. FIG. 4E depicts a gasket 445 that isconfigures to be disposed upon the cover 444 of reservoir assembly 450.Seen are three fluid transfer ports 442 for each retentate reservoir 452and for permeate/filtrate reservoir 454. Again, three pneumatic ports430, for each retentate reservoir 452 and for permeate/filtratereservoir 454, are shown.

The overall work flow for cell growth comprises loading a cell cultureto be grown into a first retentate reservoir, optionally bubbling air oran appropriate gas through the cell culture, passing or flowing the cellculture through the first retentate port then tangentially through theTFF channel structure while collecting medium or buffer through one orboth of the permeate ports 406, collecting the cell culture through asecond retentate port 404 into a second retentate reservoir, optionallyadding additional or different medium to the cell culture and optionallybubbling air or gas through the cell culture, then repeating theprocess, all while measuring, e.g., the optical density of the cellculture in the retentate reservoirs continuously or at desiredintervals. Measurements of optical densities (OD) at programmed timeintervals are accomplished using a 600 nm Light Emitting Diode (LED)that has been columnated through an optic into the retentatereservoir(s) containing the growing cells. The light continues through acollection optic to the detection system which consists of a (digital)gain-controlled silicone photodiode. Generally, optical density is shownas the absolute value of the logarithm with base 10 of the powertransmission factors of an optical attenuator: OD=−log 10 (Powerout/Power in). Since OD is the measure of optical attenuation—that is,the sum of absorption, scattering, and reflection—the TFF device ODmeasurement records the overall power transmission, so as the cells growand become denser in population, the OD (the loss of signal) increases.The OD system is pre-calibrated against OD standards with these valuesstored in an on-board memory accessible by the measurement program.

In the channel structure, the membrane bifurcating the flow channelsretains the cells on one side of the membrane (the retentate side 422)and allows unwanted medium or buffer to flow across the membrane into afiltrate or permeate side (e.g., permeate member 420) of the device.Bubbling air or other appropriate gas through the cell culture bothaerates and mixes the culture to enhance cell growth. During theprocess, medium that is removed during the flow through the channelstructure is removed through the permeate/filtrate ports 406.Alternatively, cells can be grown in one reservoir with bubbling oragitation without passing the cells through the TFF channel from onereservoir to the other.

The overall work flow for cell concentration using the TFF device/moduleinvolves flowing a cell culture or cell sample tangentially through thechannel structure. As with the cell growth process, the membranebifurcating the flow channels retains the cells on one side of themembrane and allows unwanted medium or buffer to flow across themembrane into a permeate/filtrate side (e.g., permeate member 420) ofthe device. In this process, a fixed volume of cells in medium or bufferis driven through the device until the cell sample is collected into oneof the retentate ports 404, and the medium/buffer that has passedthrough the membrane is collected through one or both of thepermeate/filtrate ports 406. All types of prokaryotic and eukaryoticcells—both adherent and non-adherent cells—can be grown in the TFFdevice. Adherent cells may be grown on beads or other cell scaffoldssuspended in medium that flow through the TFF device.

The medium or buffer used to suspend the cells in the cell concentrationdevice/module may be any suitable medium or buffer for the type of cellsbeing transformed or transfected, such as LB, SOC, TPD, YPG, YPAD, MEM,DMEM, IMDM, RPMI, Hanks', PBS and Ringer's solution, where the media maybe provided in a reagent cartridge as part of a kit. For culture ofadherent cells, cells may be disposed on beads, microcarriers, or othertype of scaffold suspended in medium. Most normal mammaliantissue-derived cells—except those derived from the hematopoieticsystem—are anchorage dependent and need a surface or cell culturesupport for normal proliferation. In the rotating growth vial describedherein, microcarrier technology is leveraged. Microcarriers ofparticular use typically have a diameter of 100-300 μm and have adensity slightly greater than that of the culture medium (thusfacilitating an easy separation of cells and medium for, e.g., mediumexchange) yet the density must also be sufficiently low to allowcomplete suspension of the carriers at a minimum stirring rate in orderto avoid hydrodynamic damage to the cells. Many different types ofmicrocarriers are available, and different microcarriers are optimizedfor different types of cells. There are positively charged carriers,such as Cytodex 1 (dextran-based, GE Healthcare), DE-52(cellulose-based, Sigma-Aldrich Labware), DE-53 (cellulose-based,Sigma-Aldrich Labware), and HLX 11-170 (polystyrene-based); collagen- orECM- (extracellular matrix) coated carriers, such as Cytodex 3(dextran-based, GE Healthcare) or HyQ-sphere Pro-F 102-4(polystyrene-based, Thermo Scientific); non-charged carriers, likeHyQ-sphere P 102-4 (Thermo Scientific); or macroporous carriers based ongelatin (Cultisphere, Percell Biolytica) or cellulose (Cytopore, GEHealthcare).

In both the cell growth and concentration processes, passing the cellsample through the TFF device and collecting the cells in one of theretentate ports 404 while collecting the medium in one of thepermeate/filtrate ports 406 is considered “one pass” of the cell sample.The transfer between retentate reservoirs “flips” the culture. Theretentate and permeatee ports collecting the cells and medium,respectively, for a given pass reside on the same end of TFFdevice/module with fluidic connections arranged so that there are twodistinct flow layers for the retentate and permeate/filtrate sides, butif the retentate port 404 resides on the retentate member ofdevice/module (that is, the cells are driven through the channel abovethe membrane and the filtrate (medium) passes to the portion of thechannel below the membrane), the permeate/filtrate port 406 will resideon the permeate member of device/module and vice versa (that is, if thecell sample is driven through the channel below the membrane, thefiltrate (medium) passes to the portion of the channel above themembrane). Due to the high pressures used to transfer the cell cultureand fluids through the flow channel of the TFF device, the effect ofgravity is negligible.

At the conclusion of a “pass” in either of the growth and concentrationprocesses, the cell sample is collected by passing through the retentateport 404 and into the retentate reservoir (not shown). To initiateanother “pass”, the cell sample is passed again through the TFF device,this time in a flow direction that is reversed from the first pass. Thecell sample is collected by passing through the retentate port 404 andinto retentate reservoir (not shown) on the opposite end of thedevice/module from the retentate port 404 that was used to collect cellsduring the first pass. Likewise, the medium/buffer that passes throughthe membrane on the second pass is collected through the permeate port406 on the opposite end of the device/module from the permeate port 406that was used to collect the filtrate during the first pass, or throughboth ports. This alternating process of passing the retentate (theconcentrated cell sample) through the device/module is repeated untilthe cells have been grown to a desired optical density, and/orconcentrated to a desired volume, and both permeate ports (i.e., ifthere are more than one) can be open during the passes to reduceoperating time. In addition, buffer exchange may be effected by adding adesired buffer (or fresh medium) to the cell sample in the retentatereservoir, before initiating another “pass”, and repeating this processuntil the old medium or buffer is diluted and filtered out and the cellsreside in fresh medium or buffer. Note that buffer exchange and cellgrowth may (and typically do) take place simultaneously, and bufferexchange and cell concentration may (and typically do) take placesimultaneously. For further information and alternative embodiments onTFFs see, e.g., U.S. Ser. No. 16/516,701, filed 5 Sep. 2019.

The Cell Transformation Module

FIG. 5A depicts a partially assembled reagent cartridge 500, comprisinga cover 501, reservoirs 504, a reservoir cover 505 comprising an outerflange 507 with an inner flange (not shown), that engages with anindentation (indentations 509 seen with uncovered reservoirs 504). Outerflange 507 provides a grip for handling the reservoir cover 505 or otherinserts for the reservoirs described below. Reservoirs 504 in reagentcartridge 500 in FIG. 5A accommodate strips of co-joined tubes,including as shown here a strip of co-joined large tubes 503 a (withindividual tubes 561). The reservoirs 504 in the embodiment of thereagent cartridge 500 shown in FIG. 5A are “slot”—shaped and areconfigured to be “universal”: that is, the slot-shaped reservoirs 504are configured to accommodate many different types of reservoir inserts.The strip of co-joined large tubes 503 a comprises an outer flange 507with an inner flange (not shown), configured to engage with anindentation 509 in the cover 501 of the reagent cartridge 500, as wellas a tab 517 configured to engage with tab engagement member 511 incover 501. Any or all reservoir inserts may be covered by a protectivefoil, film or peel-off strip to maintain sterility of the reservoir andthe contents thereof (not shown). In addition, in certain embodimentsthe material used to fabricate the cartridge is thermally-conductive, asin certain embodiments the cartridge 500 contacts a thermal device (notshown), such as a Peltier device or thermoelectric cooler, that heats orcools reagents in the reagent reservoirs or reservoirs 504.

Also shown in FIG. 5A is an insert 508 comprising a flow-throughelectroporation device 506. The flow-through electroporation device 506is configured to transform or transfect nucleic acids or other materialsinto cells and is described in detail in relation to FIGS. 5B-5F. Theflow-through electroporation device insert 508 comprises both a tab 517,and an outer flange 507. As with the strip of co-joined large tubes 503a, the outer flange 507 comprises an inner flange (not shown),configured to engage with an indentation 509 in the cover 501 of thereagent cartridge 500, as well as a tab 517 configured to engage withtab engagement member 511 in cover 501.

Reagents such as cell samples, enzymes, buffers, nucleic acid vectors,expression cassettes, proteins or peptides, reaction components (suchas, e.g., MgCl₂, dNTPs, nucleic acid assembly reagents, gap repairreagents, and the like), wash solutions, ethanol, and magnetic beads fornucleic acid purification and isolation, etc. may be positioned in thereagent cartridge at a known position. In some embodiments of cartridge500, the cartridge comprises a script (not shown) readable by aprocessor (not shown) for dispensing the reagents. Also, the cartridge500 as one component in an automated multi-module cell processinginstrument may comprise a script specifying two, three, four, five, tenor more processes to be performed by the automated multi-module cellprocessing instrument. In certain embodiments, the reagent cartridge isdisposable and is pre-packaged with reagents tailored to performingspecific cell processing protocols, e.g., genome editing or proteinproduction. Because the reagent cartridge contents vary whilecomponents/modules of the automated multi-module cell processinginstrument or system may not, the script associated with a particularreagent cartridge matches the reagents used and cell processesperformed. Thus, e.g., reagent cartridges may be pre-packaged withreagents for genome editing and a script that specifies the processsteps for performing genome editing in an automated multi-module cellprocessing instrument, or, e.g., reagents for protein expression and ascript that specifies the process steps for performing proteinexpression in an automated multi-module cell processing instrument.

For example, the reagent cartridge may comprise a script to pipettecompetent cells from a reservoir, transfer the cells to a transformationmodule, pipette a nucleic acid solution comprising a vector withexpression cassette from another reservoir in the reagent cartridge,transfer the nucleic acid solution to the transformation module,initiate the transformation process for a specified time, then move thetransformed cells to yet another reservoir in the reagent cassette or toanother module such as a cell growth module in the automatedmulti-module cell processing instrument. In another example, the reagentcartridge may comprise a script to transfer a nucleic acid solutioncomprising a vector from a reservoir in the reagent cassette, nucleicacid solution comprising editing oligonucleotide cassettes in areservoir in the reagent cassette, and a nucleic acid assembly mix fromanother reservoir to the nucleic acid assembly/desalting module, ifpresent. The script may also specify process steps performed by othermodules in the automated multi-module cell processing instrument. Forexample, the script may specify that the nucleic acid assembly/desaltingreservoir be heated to 50° C. for 30 min to generate an assembledproduct; and desalting and resuspension of the assembled product viamagnetic bead-based nucleic acid purification involving a series ofpipette transfers and mixing of magnetic beads, ethanol wash, andbuffer.

As described in relation to FIGS. 5B and 5C below, the exemplary reagentcartridges for use in the automated multi-module cell processinginstruments may include one or more electroporation devices, preferablyflow-through electroporation (FTEP) devices. In yet other embodiments,the reagent cartridge is separate from the transformation module.Electroporation is a widely-used method for permeabilization of cellmembranes that works by temporarily generating pores in the cellmembranes with electrical stimulation. Applications of electroporationinclude the delivery of DNA, RNA, siRNA, peptides, proteins, antibodies,drugs or other substances to a variety of cells such as mammalian cells(including human cells), plant cells, archea, yeasts, other eukaryoticcells, bacteria, and other cell types. Electrical stimulation may alsobe used for cell fusion in the production of hybridomas or other fusedcells. During a typical electroporation procedure, cells are suspendedin a buffer or medium that is favorable for cell survival. For bacterialcell electroporation, low conductance mediums, such as water, glycerolsolutions and the like, are often used to reduce the heat production bytransient high current. In traditional electroporation devices, thecells and material to be electroporated into the cells (collectively“the cell sample”) are placed in a cuvette embedded with two flatelectrodes for electrical discharge. For example, Bio-Rad (Hercules,Calif.) makes the GENE PULSER XCELL™ line of products to electroporatecells in cuvettes. Traditionally, electroporation requires high fieldstrength; however, the flow-through electroporation devices included inthe reagent cartridges achieve high efficiency cell electroporation withlow toxicity. The reagent cartridges of the disclosure allow forparticularly easy integration with robotic liquid handlinginstrumentation that is typically used in automated instruments andsystems such as air displacement pipettors. Such automatedinstrumentation includes, but is not limited to, off-the-shelf automatedliquid handling systems from Tecan (Mannedorf, Switzerland), Hamilton(Reno, Nev.), Beckman Coulter (Fort Collins, Colo.), etc.

FIGS. 5B and 5C are top perspective and bottom perspective views,respectively, of an exemplary FTEP device 550 that may be part of (e.g.,a component in) reagent cartridge 500 in FIG. 5A or may be a stand-alonemodule; that is, not a part of a reagent cartridge or other module. FIG.5B depicts an FTEP device 550. The FTEP device 550 has wells that definecell sample inlets 552 and cell sample outlets 554. FIG. 5C is a bottomperspective view of the FTEP device 550 of FIG. 5B. An inlet well 552and an outlet well 554 can be seen in this view. Also seen in FIG. 5Care the bottom of an inlet 562 corresponding to well 552, the bottom ofan outlet 564 corresponding to the outlet well 554, the bottom of adefined flow channel 566 and the bottom of two electrodes 568 on eitherside of flow channel 566. The FTEP devices may comprise push-pullpneumatic means to allow multi-pass electroporation procedures; that is,cells to electroporated may be “pulled” from the inlet toward the outletfor one pass of electroporation, then be “pushed” from the outlet end ofthe FTEP device toward the inlet end to pass between the electrodesagain for another pass of electroporation. Further, this process may berepeated one to many times. For additional information regarding FTEPdevices, see, e.g., U.S. Pat. No. 10,435,713, issued 8 Oct. 2019; U.S.Pat. No. 10,443,074, issued 15 Oct. 2019; U.S. Pat. No. 10,323,258,issued 18 Jun. 2019; U.S. Pat. No. 10,568,288, issued 17 Dec. 2019; andU.S. Pat. No. 10,415,058, issued 17 Sep. 2019. Further, otherembodiments of the reagent cartridge may provide or accommodateelectroporation devices that are not configured as FTEP devices, such asthose described in U.S. Ser. No. 16/109,156, filed 22 Aug. 2018. Forreagent cartridges useful in the present automated multi-module cellprocessing instruments, see, e.g., U.S. Pat. No. 10,376,889, issued 13Aug. 2019; U.S. Pat. No. 10,406,525, issued 10 Sep. 2019; U.S. Pat. No.10,478,822, issued 19 Nov. 2019; and U.S. Ser. No. 16/596,940, filed 9Oct. 2019.

Additional details of the FTEP devices are illustrated in FIGS. 5D-5F.Note that in the FTEP devices in FIGS. 5D-5F the electrodes are placedsuch that a first electrode is placed between an inlet and a narrowedregion of the flow channel, and the second electrode is placed betweenthe narrowed region of the flow channel and an outlet. FIG. 5D shows atop planar view of an FTEP device 550 having an inlet 552 forintroducing a fluid containing cells and exogenous material into FTEPdevice 550 and an outlet 554 for removing the transformed cells from theFTEP following electroporation. The electrodes 568 are introducedthrough channels (not shown) in the device. FIG. 5E shows a cutaway viewfrom the top of the FTEP device 550, with the inlet 552, outlet 554, andelectrodes 568 positioned with respect to a flow channel 566. FIG. 5Fshows a side cutaway view of FTEP device 550 with the inlet 552 andinlet channel 572, and outlet 554 and outlet channel 574. The electrodes568 are positioned in electrode channels 576 so that they are in fluidcommunication with the flow channel 566, but not directly in the path ofthe cells traveling through the flow channel 566. Note that the firstelectrode is placed between the inlet and the narrowed region of theflow channel, and the second electrode is placed between the narrowedregion of the flow channel and the outlet. The electrodes 568 in thisaspect of the device are positioned in the electrode channels 576 whichare generally perpendicular to the flow channel 566 such that the fluidcontaining the cells and exogenous material flows from the inlet channel572 through the flow channel 566 to the outlet channel 574, and in theprocess fluid flows into the electrode channels 376 to be in contactwith the electrodes 568. In this aspect, the inlet channel, outletchannel and electrode channels all originate from the same planar sideof the device. In certain aspects, however, the electrodes may beintroduced from a different planar side of the FTEP device than theinlet and outlet channels.

In the FTEP devices of the disclosure, the toxicity level of thetransformation results in greater than 30% viable cells afterelectroporation, preferably greater than 35%, 40%, 45%, 50%, 55%, 60%,70%, 75%, 80%, 85%, 90%, 95% or even 99% viable cells followingtransformation, depending on the cell type and the nucleic acids beingintroduced into the cells.

The housing of the FTEP device can be made from many materials dependingon whether the FTEP device is to be reused, autoclaved, or isdisposable, including stainless steel, silicon, glass, resin, polyvinylchloride, polyethylene, polyamide, polystyrene, polyethylene,polypropylene, acrylonitrile butadiene, polycarbonate,polyetheretheketone (PEEK), polysulfone and polyurethane, co-polymers ofthese and other polymers. Similarly, the walls of the channels in thedevice can be made of any suitable material including silicone, resin,glass, glass fiber, polyvinyl chloride, polyethylene, polyamide,polyethylene, polypropylene, acrylonitrile butadiene, polycarbonate,polyetheretheketone (PEEK), polysulfone and polyurethane, co-polymers ofthese and other polymers. Preferred materials include crystal styrene,cyclo-olefin polymer (COP) and cyclic olephin co-polymers (COC), whichallow the device to be formed entirely by injection molding in one piecewith the exception of the electrodes and, e.g., a bottom sealing film ifpresent.

The FTEP devices described herein (or portions of the FTEP devices) canbe created or fabricated via various techniques, e.g., as entire devicesor by creation of structural layers that are fused or otherwise coupled.For example, for metal FTEP devices, fabrication may include precisionmechanical machining or laser machining; for silicon FTEP devices,fabrication may include dry or wet etching; for glass FTEP devices,fabrication may include dry or wet etching, powderblasting,sandblasting, or photostructuring; and for plastic FTEP devicesfabrication may include thermoforming, injection molding, hot embossing,or laser machining. The components of the FTEP devices may bemanufactured separately and then assembled, or certain components of theFTEP devices (or even the entire FTEP device except for the electrodes)may be manufactured (e.g., using 3D printing) or molded (e.g., usinginjection molding) as a single entity, with other components added aftermolding. For example, housing and channels may be manufactured or moldedas a single entity, with the electrodes later added to form the FTEPunit. Alternatively, the FTEP device may also be formed in two or moreparallel layers, e.g., a layer with the horizontal channel and filter, alayer with the vertical channels, and a layer with the inlet and outletports, which are manufactured and/or molded individually and assembledfollowing manufacture.

In specific aspects, the FTEP device can be manufactured using a circuitboard as a base, with the electrodes, filter and/or the flow channelformed in the desired configuration on the circuit board, and theremaining housing of the device containing, e.g., the one or more inletand outlet channels and/or the flow channel formed as a separate layerthat is then sealed onto the circuit board. The sealing of the top ofthe housing onto the circuit board provides the desired configuration ofthe different elements of the FTEP devices of the disclosure. Also, twoto many FTEP devices may be manufactured on a single substrate, thenseparated from one another thereafter or used in parallel. In certainembodiments, the FTEP devices are reusable and, in some embodiments, theFTEP devices are disposable. In additional embodiments, the FTEP devicesmay be autoclavable.

The electrodes 408 can be formed from any suitable metal, such ascopper, stainless steel, titanium, aluminum, brass, silver, rhodium,gold or platinum, or graphite. One preferred electrode material is alloy303 (UNS330300) austenitic stainless steel. An applied electric fieldcan destroy electrodes made from of metals like aluminum. If amultiple-use (i.e., non-disposable) flow-through FTEP device isdesired—as opposed to a disposable, one-use flow-through FTEP device—theelectrode plates can be coated with metals resistant to electrochemicalcorrosion. Conductive coatings like noble metals, e.g., gold, can beused to protect the electrode plates.

As mentioned, the FTEP devices may comprise push-pull pneumatic means toallow multi-pass electroporation procedures; that is, cells toelectroporated may be “pulled” from the inlet toward the outlet for onepass of electroporation, then be “pushed” from the outlet end of theflow-through FTEP device toward the inlet end to pass between theelectrodes again for another pass of electroporation. This process maybe repeated one to many times.

Depending on the type of cells to be electroporated (e.g., bacterial,yeast, mammalian) and the configuration of the electrodes, the distancebetween the electrodes in the flow channel can vary widely. For example,where the flow channel decreases in width, the flow channel may narrowto between 10 μm and 5 mm, or between 25 μm and 3 mm, or between 50 μmand 2 mm, or between 75 μm and 1 mm. The distance between the electrodesin the flow channel may be between 1 mm and 10 mm, or between 2 mm and 8mm, or between 3 mm and 7 mm, or between 4 mm and 6 mm. The overall sizeof the FTEP device may be from 3 cm to 15 cm in length, or 4 cm to 12 cmin length, or 4.5 cm to 10 cm in length. The overall width of the FTEPdevice may be from 0.5 cm to 5 cm, or from 0.75 cm to 3 cm, or from 1 cmto 2.5 cm, or from 1 cm to 1.5 cm.

The region of the flow channel that is narrowed is wide enough so thatat least two cells can fit in the narrowed portion side-by-side. Forexample, a typical bacterial cell is 1 μm in diameter; thus, thenarrowed portion of the flow channel of the FTEP device used totransform such bacterial cells will be at least 2 μm wide. In anotherexample, if a mammalian cell is approximately 50 μm in diameter, thenarrowed portion of the flow channel of the FTEP device used totransform such mammalian cells will be at least 100 μm wide. That is,the narrowed portion of the FTEP device will not physically contort or“squeeze” the cells being transformed.

In embodiments of the FTEP device where reservoirs are used to introducecells and exogenous material into the FTEP device, the reservoirs rangein volume from 100 μL to 10 mL, or from 500 μL to 75 mL, or from 1 mL to5 mL. The flow rate in the FTEP ranges from 0.1 mL to 5 mL per minute,or from 0.5 mL to 3 mL per minute, or from 1.0 mL to 2.5 mL per minute.The pressure in the FTEP device ranges from 1-30 psi, or from 2-10 psi,or from 3-5 psi.

To avoid different field intensities between the electrodes, theelectrodes should be arranged in parallel. Furthermore, the surface ofthe electrodes should be as smooth as possible without pin holes orpeaks. Electrodes having a roughness Rz of 1 to 10 μm are preferred. Inanother embodiment of the invention, the flow-through electroporationdevice comprises at least one additional electrode which applies aground potential to the FTEP device. Flow-through electroporationdevices (either as a stand-alone instrument or as a module in anautomated multi-module system) are described in, e.g., U.S. Ser. No.16/147,120, filed 28 Sep. 2018; Ser. No. 16/147,353, filed 28 Sep. 2018;Ser. No. 16/147,865, filed 30 Sep. 2018; and Ser. No. 16/426,310, filed30 May 2019; and U.S. Pat. No. 10,323,258, issued 18 Jun. 2019.

Cell Singulation and Enrichment Device

FIG. 6A depicts a solid wall device 6050 and a workflow for singulatingcells in microwells in the solid wall device. At the top left of thefigure (i), there is depicted solid wall device 6050 with microwells6052. A section 6054 of substrate 6050 is shown at (ii), also depictingmicrowells 6052. At (iii), a side cross-section of solid wall device6050 is shown, and microwells 6052 have been loaded, where, in thisembodiment, Poisson or substantial Poisson loading has taken place; thatis, each microwell has one or no cells, and the likelihood that any onemicrowell has more than one cell is low. At (iv), workflow 6040 isillustrated where substrate 6050 having microwells 6052 shows microwells6056 with one cell per microwell, microwells 6057 with no cells in themicrowells, and one microwell 6060 with two cells in the microwell. Instep 6051, the cells in the microwells are allowed to doubleapproximately 2-150 times to form clonal colonies (v), then editing isallowed to occur 6053.

After editing 6053, many cells in the colonies of cells that have beenedited die as a result of the double-strand cuts caused by activeediting and there is a lag in growth for the edited cells that dosurvive but must repair and recover following editing (microwells 6058),where cells that do not undergo editing thrive (microwells 6059) (vi).All cells are allowed to continue grow to establish colonies andnormalize, where the colonies of edited cells in microwells 6058 catchup in size and/or cell number with the cells in microwells 6059 that donot undergo editing (vii). Once the cell colonies are normalized, eitherpooling 6060 of all cells in the microwells can take place, in whichcase the cells are enriched for edited cells by eliminating the biasfrom non-editing cells and fitness effects from editing; alternatively,colony growth in the microwells is monitored after editing, and slowgrowing colonies (e.g., the cells in microwells 6058) are identified andselected 6061 (e.g., “cherry picked”) resulting in even greaterenrichment of edited cells.

In growing the cells, the medium used will depend, of course, on thetype of cells being edited—e.g., bacterial, yeast or mammalian. Forexample, medium for yeast cell growth includes LB, SOC, TPD, YPG, YPAD,MEM and DMEM.

FIG. 6B depicts a solid wall device 6050 and a workflow forsubstantially singulating cells in microwells in a solid wall device. Atthe top left of the figure (i), there is depicted solid wall device 6050with microwells 6052. A section 6054 of substrate 6050 is shown at (ii),also depicting microwells 6052. At (iii), a side cross-section of solidwall device 6050 is shown, and microwells 6052 have been loaded, where,in this embodiment, substantial Poisson loading has taken place; thatis, some microwells 6057 have no cells, and some microwells 6076, 6078have a few cells. In FIG. 6B, cells with active gRNAs are shown as solidcircles, and cells with inactive gRNAs are shown as open circles. At(iv), workflow 6070 is illustrated where substrate 6050 havingmicrowells 6052 shows three microwells 6076 with several cells all withactive gRNAs, microwell 6057 with no cells, and two microwells 6078 withsome cells having active gRNAs and some cells having inactive gRNAs. Instep 6071, the cells in the microwells are allowed to doubleapproximately 2-150 times to form clonal colonies (v), then editingtakes place 6073.

After editing 6073, many cells in the colonies of cells that have beenedited die as a result of the double-strand cuts caused by activeediting and there is a lag in growth for the edited cells that dosurvive but must repair and recover following editing (microwells 6076),where cells that do not undergo editing thrive (microwells 6078) (vi).Thus, in microwells 6076 where only cells with active gRNAs reside(cells depicted by solid circles), most cells die off; however, inmicrowells 6078 containing cells with inactive gRNAs (cells depicted byopen circles), cells continue to grow and are not impacted by activeediting. The cells in each microwell (6076 and 6078) are allowed to growto continue to establish colonies and normalize, where the colonies ofedited cells in microwells 6076 catch up in size and/or cell number withthe unedited cells in microwells 6078 that do not undergo editing (vii).Note that in this workflow 6070, the colonies of cells in the microwellsare not clonal; that is, not all cells in a well arise from a singlecell. Instead, the cell colonies in the well may be mixed colonies,arising in many wells from two to several different cells. Once the cellcolonies are normalized, either pooling 6090 of all cells in themicrowells can take place, in which case the cells are enriched foredited cells by eliminating the bias from non-editing cells and fitnesseffects from editing; alternatively, colony growth in the microwells ismonitored after editing, and slow growing colonies (e.g., the cells inmicrowells 6076) are identified and selected 6091 (e.g., “cherrypicked”) resulting in even greater enrichment of edited cells.

A module useful for performing the methods depicted in FIGS. 6A and 6Bis a solid wall isolation, incubation, and normalization (SWIIN) module.FIG. 6C depicts an embodiment of a SWIIN module 650 from an exploded topperspective view. In SWIIN module 650 the retentate member is formed onthe bottom of a top of a SWIIN module component and the permeate memberis formed on the top of the bottom of a SWIIN module component.

The SWIIN module 650 in FIG. 6C comprises from the top down, a reservoirgasket or cover 658, a retentate member 604 (where a retentate flowchannel cannot be seen in this FIG. 6C), a perforated member 601 swagedwith a filter (filter not seen in FIG. 6C), a permeate member 608comprising integrated reservoirs (permeate reservoirs 652 and retentatereservoirs 654), and two reservoir seals 662, which seal the bottom ofpermeate reservoirs 652 and retentate reservoirs 654. A permeate channel660 a can be seen disposed on the top of permeate member 608, defined bya raised portion 676 of serpentine channel 660 a, and ultrasonic tabs664 can be seen disposed on the top of permeate member 608 as well. Theperforations that form the wells on perforated member 601 are not seenin this FIG. 6C; however, through-holes 666 to accommodate theultrasonic tabs 664 are seen. In addition, supports 670 are disposed ateither end of SWIIN module 650 to support SWIIN module 650 and toelevate permeate member 608 and retentate member 604 above reservoirs652 and 654 to minimize bubbles or air entering the fluid path from thepermeate reservoir to serpentine channel 660 a or the fluid path fromthe retentate reservoir to serpentine channel 660 b (neither fluid pathis seen in this FIG. 6C).

In this FIG. 6C, it can be seen that the serpentine channel 660 a thatis disposed on the top of permeate member 608 traverses permeate member608 for most of the length of permeate member 608 except for the portionof permeate member 608 that comprises permeate reservoirs 652 andretentate reservoirs 654 and for most of the width of permeate member608. As used herein with respect to the distribution channels in theretentate member or permeate member, “most of the length” means about95% of the length of the retentate member or permeate member, or about90%, 85%, 80%, 75%, or 70% of the length of the retentate member orpermeate member. As used herein with respect to the distributionchannels in the retentate member or permeate member, “most of the width”means about 95% of the width of the retentate member or permeate member,or about 90%, 85%, 80%, 75%, or 70% of the width of the retentate memberor permeate member.

In this embodiment of a SWIIN module, the perforated member includesthrough-holes to accommodate ultrasonic tabs disposed on the permeatemember. Thus, in this embodiment the perforated member is fabricatedfrom 316 stainless steel, and the perforations form the walls ofmicrowells while a filter or membrane is used to form the bottom of themicrowells. Typically, the perforations (microwells) are approximately150 μm-200 μm in diameter, and the perforated member is approximately125 μm deep, resulting in microwells having a volume of approximately2.5 nl, with a total of approximately 200,000 microwells. The distancebetween the microwells is approximately 279 μm center-to-center. Thoughhere the microwells have a volume of approximately 2.5 nl, the volume ofthe microwells may be from 1 to 25 nl, or preferably from 2 to 10 nl,and even more preferably from 2 to 4 nl. As for the filter or membrane,like the filter described previously, filters appropriate for use aresolvent resistant, contamination free during filtration, and are able toretain the types and sizes of cells of interest. For example, in orderto retain small cell types such as bacterial cells, pore sizes can be aslow as 0.10 μm, however for other cell types (e.g., such as formammalian cells), the pore sizes can be as high as 10.0 μm-20.0 μm ormore. Indeed, the pore sizes useful in the cell concentrationdevice/module include filters with sizes from 0.10 μm, 0.11 μm, 0.12 μm,0.13 μm, 0.14 μm, 0.15 μm, 0.16 μm, 0.17 μm, 0.18 μm, 0.19 μm, 0.20 μm,0.21 μm, 0.22 μm, 0.23 μm, 0.24 μm, 0.25 μm, 0.26 μm, 0.27 μm, 0.28 μm,0.29 μm, 0.30 μm, 0.31 μm, 0.32 μm, 0.33 μm, 0.34 μm, 0.35 μm, 0.36 μm,0.37 μm, 0.38 μm, 0.39 μm, 0.40 μm, 0.41 μm, 0.42 μm, 0.43 μm, 0.44 μm,0.45 μm, 0.46 μm, 0.47 μm, 0.48 μm, 0.49 μm, 0.50 μm and larger. Thefilters may be fabricated from any suitable material including cellulosemixed ester (cellulose nitrate and acetate) (CME), polycarbonate (PC),polyvinylidene fluoride (PVDF), polyethersulfone (PES),polytetrafluoroethylene (PTFE), nylon, or glass fiber.

The cross-section configuration of the mated serpentine channel may beround, elliptical, oval, square, rectangular, trapezoidal, or irregular.If square, rectangular, or another shape with generally straight sides,the cross section may be from about 2 mm to 15 mm wide, or from 3 mm to12 mm wide, or from 5 mm to 10 mm wide. If the cross section of themated serpentine channel is generally round, oval or elliptical, theradius of the channel may be from about 3 mm to 20 mm in hydraulicradius, or from 5 mm to 15 mm in hydraulic radius, or from 8 mm to 12 mmin hydraulic radius.

Serpentine channels 660 a and 660 b can have approximately the samevolume or a different volume. For example, each “side” or portion 660 a,660 b of the serpentine channel may have a volume of, e.g., 2 mL, orserpentine channel 660 a of permeate member 608 may have a volume of 2mL, and the serpentine channel 660 b of retentate member 604 may have avolume of, e.g., 3 mL. The volume of fluid in the serpentine channel mayrange from about 2 mL to about 80 mL, or about 4 mL to 60 mL, or from 5mL to 40 mL, or from 6 mL to 20 mL (note these volumes apply to a SWIINmodule comprising a, e.g., 50-500K perforation member). The volume ofthe reservoirs may range from 5 mL to 50 mL, or from 7 mL to 40 mL, orfrom 8 mL to 30 mL or from 10 mL to 20 mL, and the volumes of allreservoirs may be the same or the volumes of the reservoirs may differ(e.g., the volume of the permeate reservoirs is greater than that of theretentate reservoirs).

The serpentine channel portions 660 a and 660 b of the permeate member608 and retentate member 604, respectively, are approximately 200 mmlong, 130 mm wide, and 4 mm thick, though in other embodiments, theretentate and permeate members can be from 75 mm to 400 mm in length, orfrom 100 mm to 300 mm in length, or from 150 mm to 250 mm in length;from 50 mm to 250 mm in width, or from 75 mm to 200 mm in width, or from100 mm to 150 mm in width; and from 2 mm to 15 mm in thickness, or from4 mm to 10 mm in thickness, or from 5 mm to 8 mm in thickness.Embodiments the retentate (and permeate) members may be fabricated fromPMMA (poly(methyl methacrylate) or other materials may be used,including polycarbonate, cyclic olefin co-polymer (COC), glass,polyvinyl chloride, polyethylene, polyamide, polypropylene, polysulfone,polyurethane, and co-polymers of these and other polymers. Preferably atleast the retentate member is fabricated from a transparent material sothat the cells can be visualized (see, e.g., FIG. 6F and the descriptionthereof). For example, a video camera may be used to monitor cell growthby, e.g., density change measurements based on an image of an emptywell, with phase contrast, or if, e.g., a chromogenic marker, such as achromogenic protein, is used to add a distinguishable color to thecells. Chromogenic markers such as blitzen blue, dreidel teal, virginiaviolet, vixen purple, prancer purple, tinsel purple, maccabee purple,donner magenta, cupid pink, seraphina pink, scrooge orange, and leororange (the Chromogenic Protein Paintbox, all available from ATUM(Newark, Calif.)) obviate the need to use fluorescence, althoughfluorescent cell markers, fluorescent proteins, and chemiluminescentcell markers may also be used.

Because the retentate member preferably is transparent, colony growth inthe SWIIN module can be monitored by automated devices such as thosesold by JoVE (ScanLag™ system, Cambridge, Mass.) (also seeLevin-Reisman, et al., Nature Methods, 7:737-39 (2010)). Cell growthfor, e.g., mammalian cells may be monitored by, e.g., the growth monitorsold by IncuCyte (Ann Arbor, Mich.) (see also, Choudhry, PLos One,11(2):e0148469 (2016)). Further, automated colony pickers may beemployed, such as those sold by, e.g., TECAN (Pickolo™ system,Mannedorf, Switzerland); Hudson Inc. (RapidPick™, Springfield, N.J.);Molecular Devices (QPix 400™ system, San Jose, Calif.); and SingerInstruments (PIXL™ system, Somerset, UK).

Due to the heating and cooling of the SWIIN module, condensation mayaccumulate on the retentate member which may interfere with accuratevisualization of the growing cell colonies. Condensation of the SWIINmodule 650 may be controlled by, e.g., moving heated air over the top of(e.g., retentate member) of the SWIIN module 650, or by applying atransparent heated lid over at least the serpentine channel portion 660b of the retentate member 604. See, e.g., FIG. 6F and the descriptionthereof infra.

In SWIIN module 650 cells and medium—at a dilution appropriate forPoisson or substantial Poisson distribution of the cells in themicrowells of the perforated member—are flowed into serpentine channel660 b from ports in retentate member 604, and the cells settle in themicrowells while the medium passes through the filter into serpentinechannel 660 a in permeate member 608. The cells are retained in themicrowells of perforated member 601 as the cells cannot travel throughfilter 603. Appropriate medium may be introduced into permeate member608 through permeate ports 611. The medium flows upward through filter603 to nourish the cells in the microwells (perforations) of perforatedmember 601. Additionally, buffer exchange can be effected by cyclingmedium through the retentate and permeate members. In operation, thecells are deposited into the microwells, are grown for an initial, e.g.,2-100 doublings, editing is induced by, e.g., raising the temperature ofthe SWIIN to 42° C. to induce a temperature inducible promoter or byremoving growth medium from the permeate member and replacing the growthmedium with a medium comprising a chemical component that induces aninducible promoter.

Once editing has taken place, the temperature of the SWIIN may bedecreased, or the inducing medium may be removed and replaced with freshmedium lacking the chemical component thereby de-activating theinducible promoter. The cells then continue to grow in the SWIIN module650 until the growth of the cell colonies in the microwells isnormalized. For the normalization protocol, once the colonies arenormalized, the colonies are flushed from the microwells by applyingfluid or air pressure (or both) to the permeate member serpentinechannel 660 a and thus to filter 603 and pooled. Alternatively, ifcherry picking is desired, the growth of the cell colonies in themicrowells is monitored, and slow-growing colonies are directlyselected; or, fast-growing colonies are eliminated.

FIG. 6D is a top perspective view of a SWIIN module with the retentateand perforated members in partial cross section. In this FIG. 6D, it canbe seen that serpentine channel 660 a is disposed on the top of permeatemember 608 is defined by raised portions 676 and traverses permeatemember 608 for most of the length and width of permeate member 608except for the portion of permeate member 608 that comprises thepermeate and retentate reservoirs (note only one retentate reservoir 652can be seen). Moving from left to right, reservoir gasket 658 isdisposed upon the integrated reservoir cover 678 (cover not seen in thisFIG. 6D) of retentate member 604. Gasket 658 comprises reservoir accessapertures 632 a, 632 b, 632 c, and 632 d, as well as pneumatic ports 633a, 633 b, 633 c and 633 d. Also at the far left end is support 670.Disposed under permeate reservoir 652 can be seen one of two reservoirseals 662. In addition to the retentate member being in cross section,the perforated member 601 and filter 603 (filter 603 is not seen in thisFIG. 6D) are in cross section. Note that there are a number ofultrasonic tabs 664 disposed at the right end of SWIIN module 650 and onraised portion 676 which defines the channel turns of serpentine channel660 a, including ultrasonic tabs 664 extending through through-holes 666of perforated member 601. There is also a support 670 at the end distalreservoirs 652, 654 of permeate member 608.

FIG. 6E is a side perspective view of an assembled SWIIIN module 650,including, from right to left, reservoir gasket 658 disposed uponintegrated reservoir cover 678 (not seen) of retentate member 604.Gasket 658 may be fabricated from rubber, silicone, nitrile rubber,polytetrafluoroethylene, a plastic polymer such aspolychlorotrifluoroethylene, or other flexible, compressible material.Gasket 658 comprises reservoir access apertures 632 a, 632 b, 632 c, and632 d, as well as pneumatic ports 633 a, 633 b, 633 c and 633 d. Also atthe far-left end is support 670 of permeate member 608. In addition,permeate reservoir 652 can be seen, as well as one reservoir seal 662.At the far-right end is a second support 670.

Imaging of cell colonies growing in the wells of the SWIIN is desired inmost implementations for, e.g., monitoring both cell growth and deviceperformance and imaging is necessary for cherry-picking implementations.Real-time monitoring of cell growth in the SWIIN requires backlighting,retentate plate (top plate) condensation management and a system-levelapproach to temperature control, air flow, and thermal management. Insome implementations, imaging employs a camera or CCD device withsufficient resolution to be able to image individual wells. For example,in some configurations a camera with a 9-pixel pitch is used (that is,there are 9 pixels center-to-center for each well). Processing theimages may, in some implementations, utilize reading the images ingrayscale, rating each pixel from low to high, where wells with no cellswill be brightest (due to full or nearly-full light transmission fromthe backlight) and wells with cells will be dim (due to cells blockinglight transmission from the backlight). After processing the images,thresholding is performed to determine which pixels will be called“bright” or “dim”, spot finding is performed to find bright pixels andarrange them into blocks, and then the spots are arranged on a hexagonalgrid of pixels that correspond to the spots. Once arranged, the measureof intensity of each well is extracted, by, e.g., looking at one or morepixels in the middle of the spot, looking at several to many pixels atrandom or pre-set positions, or averaging X number of pixels in thespot. In addition, background intensity may be subtracted. Thresholdingis again used to call each well positive (e.g., containing cells) ornegative (e.g., no cells in the well). The imaging information may beused in several ways, including taking images at time points formonitoring cell growth. Monitoring cell growth can be used to, e.g.,remove the “muffin tops” of fast-growing cells followed by removal ofall cells or removal of cells in “rounds” as described above, or recovercells from specific wells (e.g., slow-growing cell colonies);alternatively, wells containing fast-growing cells can be identified andareas of UV light covering the fast-growing cell colonies can beprojected (or rastered with shutters) onto the SWIIN to irradiate orinhibit growth of those cells. Imaging may also be used to assure properfluid flow in the serpentine channel 660.

FIG. 6F depicts the embodiment of the SWIIN module in FIGS. 6A-6Efurther comprising a heat management system including a heater and aheated cover. The heater cover facilitates the condensation managementthat is required for imaging. Assembly 698 comprises a SWIIN module 650seen lengthwise in cross section, where one permeate reservoir 652 isseen. Disposed immediately upon SWIIN module 650 is cover 694 anddisposed immediately below SWIIN module 650 is backlight 680, whichallows for imaging. Beneath and adjacent to the backlight and SWIINmodule is insulation 682, which is disposed over a heatsink 684. In thisFIG. 6F, the fins of the heatsink would be in-out of the page. Inaddition there is also axial fan 686 and heat sink 688, as well as twothermoelectric coolers 692, and a controller 690 to control thepneumatics, thermoelectric coolers, fan, solenoid valves, etc. Thearrows denote cool air coming into the unit and hot air being removedfrom the unit. It should be noted that control of heating allows forgrowth of many different types of cells (prokaryotic and eukaryotic) aswell as strains of cells that are, e.g., temperature sensitive, etc.,and allows use of temperature-sensitive promoters. Temperature controlallows for protocols to be adjusted to account for differences intransformation efficiency, cell growth and viability. For more detailsregarding solid wall isolation incubation and normalization devices seeU.S. Pat. No. 10,533,152, issued 14 Jan. 2020; and U.S. Pat. No.10,550,363, issued 4 Feb. 2020; and U.S. Ser. No. 16/597,826, filed 19Oct. 2019; Ser. No. 16/597,831, filed 9 Oct. 2019; and Ser. No.16/693,630, filed 25 Nov. 2019. For alternative isolation, incubationand normalization modules, see U.S. Pat. No. 10,532,324, issued 14 Jan.2020; and U.S. Ser. No. 16/687,640, filed 18 Nov. 2019; and Ser. No.16/686,066, filed 15 Nov. 2019.

Use of the Automated Multi-Module Cell Processing Instrument

One embodiment of an automated multi-module cell processing instrumentcapable of performing the methods described herein is shown in FIG. 7.FIG. 7 illustrates another embodiment of a multi-module cell processinginstrument 700. This embodiment depicts an exemplary system thatperforms recursive gene editing on a cell population. The cellprocessing instrument 700 may include a housing 744, a reservoir forstoring cells to be transformed or transfected 702, and a cell growthmodule (comprising, e.g., a rotating growth vial) 704. The cells to betransformed are transferred from a reservoir to the cell growth module704 to be cultured until the cells hit a target OD. Once the cells hitthe target OD, the growth module may cool or freeze the cells for laterprocessing or transfer the cells to a cell concentration module 760where the cells are subjected to buffer exchange and renderedelectrocompetent, and the volume of the cells may be reducedsubstantially. Once the cells have been concentrated to an appropriatevolume, the cells are transferred to electroporation device or module708. In addition to the reservoir for storing cells, the multi-modulecell processing instrument 700 includes a reservoir for storing thevector pre-assembled with editing oligonucleotide cassettes 752. Thepre-assembled nucleic acid vectors are transferred to theelectroporation device 708, which already contains the cell culturegrown to a target OD. In the electroporation device 708, the nucleicacids are electroporated into the cells. Following electroporation, thecells are transferred into an optional recovery (and optionally,dilution) module 756, where the cells are allowed to recover brieflypost-transformation.

After recovery, the cells may be transferred to a storage module 712,where the cells can be stored at, e.g., 4° C. for later processing, orthe cells may be diluted and transferred to aselection/growth/induction/editing module 758. The cells are allowed togrow and editing is then induced by providing conditions (e.g.,temperature, addition of an inducing or repressing chemical) to induceediting. Note that the selection/growth/induction and editing modulesmay be the same module or device, where all processes are performed in,e.g., a solid wall singulation device, or selection and/or dilution maytake place in a separate vessel before the cells are transferred to aninduction/editing module. As an alternative to singulation in, e.g., asolid wall device, the transformed cells may be grown in—and editing canbe induced in—bulk liquid (see, e.g., U.S. Ser. No. 16/545,097, filed 20Aug. 2019. Once the putatively-edited cells are pooled, they may besubjected to another round of editing, beginning with growth, cellconcentration and treatment to render electrocompetent, andtransformation by yet another donor nucleic acid in another editingcassette via the electroporation device/module 708.

In electroporation device 708, the cells selected from the first roundof editing are transformed by a second set of editing oligos (or othertype of oligos) and the cycle is repeated until the cells have beentransformed and edited by a desired number of, e.g., editing cassettes.The multi-module cell processing instrument 700 exemplified in FIG. 7 iscontrolled by a processor 742 configured to operate the instrument basedon user input or is controlled by one or more scripts including at leastone script associated with the reagent cartridge. The processor 742 maycontrol the timing, duration, and temperature of various processes, thedispensing of reagents, and other operations of the various modules ofthe instrument 700. For example, a script or the processor may controlthe dispensing of cells, reagents, vectors, and editingoligonucleotides; which editing oligonucleotides are used for cellediting and in what order; the time, temperature and other conditionsused in the recovery and expression module, the wavelength at which ODis read in the cell growth module, the target OD to which the cells aregrown, and the target time at which the cells will reach the target OD.In addition, the processor may be programmed to notify a user (e.g., viaan application) as to the progress of the cells in the automatedmulti-module cell processing instrument.

It should be apparent to one of ordinary skill in the art given thepresent disclosure that the process described may be recursive andmultiplexed; that is, cells may go through the workflow described inrelation to FIG. 7, then the resulting edited culture may go throughanother (or several or many) rounds of additional editing (e.g.,recursive editing) with different editing vectors. For example, thecells from round 1 of editing may be diluted and an aliquot of theedited cells edited by editing vector A may be combined with editingvector B, an aliquot of the edited cells edited by editing vector A maybe combined with editing vector C, an aliquot of the edited cells editedby editing vector A may be combined with editing vector D, and so on fora second round of editing. After round two, an aliquot of each of thedouble-edited cells may be subjected to a third round of editing, where,e.g., aliquots of each of the AB-, AC-, AD-edited cells are combinedwith additional editing vectors, such as editing vectors X, Y, and Z.That is that double-edited cells AB may be combined with and edited byvectors X, Y, and Z to produce triple-edited edited cells ABX, ABY, andABZ; double-edited cells AC may be combined with and edited by vectorsX, Y, and Z to produce triple-edited cells ACX, ACY, and ACZ; anddouble-edited cells AD may be combined with and edited by vectors X, Y,and Z to produce triple-edited cells ADX, ADY, and ADZ, and so on. Inthis process, many permutations and combinations of edits can beexecuted, leading to very diverse cell populations and cell libraries.In any recursive process, it is advantageous to “cure” the previousengine and editing vectors (or single engine+editing vector in a singlevector system). “Curing” is a process in which one or more vectors usedin the prior round of editing is eliminated from the transformed cells.Curing can be accomplished by, e.g., cleaving the vector(s) using acuring plasmid thereby rendering the editing and/or engine vector (orsingle, combined vector) nonfunctional; diluting the vector(s) in thecell population via cell growth (that is, the more growth cycles thecells go through, the fewer daughter cells will retain the editing orengine vector(s)), or by, e.g., utilizing a heat-sensitive origin ofreplication on the editing or engine vector (or combined engine+editingvector). The conditions for curing will depend on the mechanism used forcuring; that is, in this example, how the curing plasmid cleaves theediting and/or engine plasmid.

The automated modules and reagent dispensing of the automatedmulti-module cell editing instruments are controlled by a processingsystem and processing environment, such as described with reference toFIG. 8. In FIG. 8, the processing system 810 includes a CPU 808 whichperforms a portion of the processes described above. For example, theCPU 808 may manage the processing stages of the method 100 of FIG. 1and/or the workflow of FIG. 7. The process data and, scripts,instructions, and/or user settings may be stored in memory 802. Theseprocess data and, scripts, instructions, and/or user settings may alsobe stored on a storage medium disk 804 such as a portable storage medium(e.g., USB drive, optical disk drive, etc.) or may be stored remotely.For example, the process data and, scripts, instructions, and/or usersettings may be stored in a location accessible to the processing system810 via a network 828. Further, the claimed advancements are not limitedby the form of the computer-readable media on which the instructions ofthe inventive process are stored. For example, the instructions may bestored in FLASH memory, RAM, ROM, or any other information processingdevice with which the processing system 810 communicates, such as aserver, computer, smart phone, or other hand-held computing device.

Further, components of the claimed advancements may be provided as autility application, background daemon, or component of an operatingsystem, or combination thereof, executing in conjunction with CPU 808and an operating system such as with other computing systems known tothose skilled in the art.

CPU 808 may be an ARM processor, system-on-a-chip (SOC), microprocessor,microcontroller, digital signal processor (DSP), or may be otherprocessor types that would be recognized by one of ordinary skill in theart. Further, CPU 808 may be implemented as multiple processorscooperatively working in parallel to perform the instructions of theinventive processes described above.

The processing system 810 is part of a processing environment 800. Theprocessing system 810 in FIG. 8 also includes a network controller 806for interfacing with the network 828 to access additional elementswithin the processing environment 800. As can be appreciated, thenetwork 828 can be a public network, such as the Internet, or a privatenetwork such as an LAN or WAN network, or any combination thereof andcan also include PSTN or ISDN sub-networks. The network 828 can bewireless such as a cellular network including EDGE, 3G and 4G wirelesscellular systems. The wireless network can also be Wi-Fi, Bluetooth, orany other wireless form of communication that is known.

The processing system 810 further includes a general purpose I/Ointerface 812 interfacing with a user interface (e.g., touch screen)816, one or more sensors 814, and one or more peripheral devices 818.The peripheral I/O devices 818 may include, in some examples, a videorecording system, an audio recording system, microphone, externalstorage devices, and/or external speaker systems. The one or moresensors 814 may include one or more of a gyroscope, an accelerometer, agravity sensor, a linear accelerometer, a global positioning system, abar code scanner, a QR code scanner, an RFID scanner, a temperaturemonitor, and a lighting system or lighting element.

The general purpose storage controller 824 connects the storage mediumdisk 1804 with communication bus 840, such as a parallel bus or a serialbus such as a Universal Serial Bus (USB), or similar, forinterconnecting all of the components of the processing system. Adescription of the general features and functionality of the storagecontroller 824, network controller 806, and general purpose I/Ointerface 812 is omitted herein for brevity as these features are known.

The processing system 810, in some embodiments, includes one or moreonboard and/or peripheral sensors 814. The sensors 814, for example, canbe incorporated directly into the internal electronics and/or a housingof the automated multi-module processing instrument. A portion of thesensors 814 can be in direct physical contact with the I/O interface812, e.g., via a wire; or in wireless contact e.g., via a Bluetooth,Wi-Fi or NFC connection. For example, a wireless communicationscontroller 826 may enable communications between one or more wirelesssensors 1814 and the I/O interface 812. Furthermore, one or more sensors814 may be in indirect contact e.g., via intermediary servers or storagedevices that are based in the network 828; or in (wired, wireless orindirect) contact with a signal accumulator somewhere within theautomated multi-module cell editing instrument, which in turn is in(wired or wireless or indirect) contact with the I/O interface 812.

A group of sensors 814 communicating with the I/O interface 812 may beused in combination to gather a given signal type from multiple placesin order to generate a more complete map of signals. One or more sensors814 communicating with the I/O interface 812 can be used as a comparatoror verification element, for example to filter, cancel, or reject othersignals.

In some embodiments, the processing environment 800 includes a computingdevice 838 communicating with the processing system 810 via the wirelesscommunications controller 826. For example, the wireless communicationscontroller 826 may enable the exchange of email messages, text messages,and/or software application alerts designated to a smart phone or otherpersonal computing device of a user.

The processing environment 800, in some implementations, includes arobotic material handling system 822. The processing system 810 mayinclude a robotics controller 820 for issuing control signals to actuateelements of the robotic material handling system, such as manipulating aposition of a gantry, lowering or raising a sipper or pipettor element,and/or actuating pumps and valves to cause liquid transfer between asipper/pipettor and various vessels (e.g., chambers, vials, etc.) in theautomated multi-module cell editing instrument. The robotics controller820, in some examples, may include a hardware driver, firmware element,and/or one or more algorithms or software packages for interfacing theprocessing system 810 with the robotics material handling system 822.

In some implementations, the processing environment 810 includes one ormore module interfaces 832, such as, in some examples, one or moresensor interfaces, power control interfaces, valve and pump interfaces,and/or actuator interfaces for activating and controlling processing ofeach module of the automated multi-module processing system. Forexample, the module interfaces 832 may include an actuator interface forthe drive motor of rotating cell growth device 200 (FIG. 3A) and asensor interface for a detector board that senses optical density ofcell growth within the rotating growth vial. A module controller 830, insome embodiments, is configured to interface with the module interfaces832. The module controller 830 may include one or many controllers(e.g., possibly one controller per module, although some modules mayshare a single controller). The module controller 830, in some examples,may include a hardware driver, firmware element, and/or one or morealgorithms or software packages for interfacing the processing system810 with the module interfaces 832.

The processing environment 810, in some implementations, includes athermal management system 836 for controlling climate conditions withinthe housing of the automated multi-module processing system. The thermalmanagement system 836 may additional control climate conditions withinone or more modules of the automated multi-module cell editinginstrument. The processing system 810, in some embodiments, includes atemperature controller 834 for interfacing with the thermal managementsystem 836. The temperature controller 834, in some examples, mayinclude a hardware driver, firmware element, and/or one or morealgorithms or software packages for interfacing the processing system810 with the thermal management system 836.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention, nor are theyintended to represent or imply that the experiments below are all of orthe only experiments performed. It will be appreciated by personsskilled in the art that numerous variations and/or modifications may bemade to the invention as shown in the specific aspects without departingfrom the spirit or scope of the invention as broadly described. Thepresent aspects are, therefore, to be considered in all respects asillustrative and not restrictive.

Example #1

Cloning Target Polynucleotide The FtsY gene from Bacillus subtilis wasamplified by PCR from a source plasmid template pJet1.2 FtsY (Addgenecatalog #117130) and cloned into the polylinker sequence of the pDG1662,a Bacillus subtilis integration vector, by restriction enzyme digest andconventional sticky end cloning. pDG1662 contains the cat gene whichconfers resistance to chloramphenicol; the bla gene which confersampicillin resistance for selection in E. coli; the spc gene whichconfers spectinomycin resistance for selection in E. coli or B.subtilis; two regions of homology to the B. subtilis amyE locus,upstream and downstream, respectively, which mediate ectopic integrationof the cloned target polynucleotide into the B. subtilis genome as adouble-crossover recombination at the amyE locus; and a replicationorigin active in E. coli but not in B. subtilis, to guarantee that onlyB. subtilis clones with successful chromosomal integrations propagateafter transformation and selection.

Ligation of the amplified target nucleotide was performed in NEB 5-alphacompetent E. coli cells according to standard conditions andtransformants were grown on ampicillin plates (100m/mL). Positive cloneswere isolated and successful insertion of the FtsY gene was confirmed bydiagnostic restriction enzyme digest and colony PCR across the insertjunctions. The resulting E. coli strain carrying the FtsY shuttle vectorwas designated “FtsY shuttle vector” cells.

Editing Cassette and Backbone Amplification and Assembly

Editing Cassette Preparation: 5 nM of oligonucleotides directed towardsaturation mutagenesis of the FtsY gene synthesized on a chip wereamplified using Q5 polymerase in 50 μL volumes. The PCR conditions were95° C. for 1 minute; 8 rounds of 95° C. for 30 seconds/60° C. for 30seconds/72° C. for 2.5 minutes; with a final hold at 72° C. for 5minutes. Following amplification, the PCR products were subjected toSPRI cleanup, where 30 μL SPRI mix was added to the 50 μL PCR reactionsand incubated for 2 minutes. The tubes were subjected to a magneticfield for 2 minutes, the liquid was removed, and the beads were washed2× with 80% ethanol, allowing 1 minute between washes. After the finalwash, the beads were allowed to dry for 2 minutes, 50 μL 0.5× TE pH 8.0was added to the tubes, and the beads were vortexed to mix. The slurrywas incubated at room temperature for 2 minutes, then subjected to themagnetic field for 2 minutes. The eluate was removed, and the DNAquantified.

Following quantification, a second amplification procedure was carriedout using a dilution of the eluate from the SPRI cleanup. PCR wasperformed under the following conditions: 95° C. for 1 minute; 18 roundsof 95° C. for 30 seconds/72° C. for 2.5 minutes; with a final hold at72° C. for 5 minutes. Amplicons were checked on a 2% agarose gel andpools with the cleanest output(s) were identified. Amplificationproducts appearing to have heterodimers or chimeras were not used.

Backbone Preparation: A 10-fold serial dilution series of purifiedbackbone was performed, and each of the diluted backbone series wasamplified under the following conditions: 95° C. for 1 minute; then 30rounds of 95° C. for 30 seconds/60° C. for 1.5 minutes/72° C. for 2.5minutes; with a final hold at 72° C. for 5 minutes. After amplification,the amplified backbone was subjected to SPRI cleanup as described abovein relation to the cassettes. The backbone was eluted into 100 μL ddH₂Oand quantified before Gibson Assembly®.

Isothermal Assembly: 150 ng backbone DNA was combined with 100 ngcassette DNA. An equal volume of 2× Master Mix was added, and thereaction was incubated for 45 minutes at 50° C. After assembly, theassembled backbone and cassettes were subjected to SPRI cleanup, asdescribed above.

Transformation with Engine Vector: 1 μL of the engine vector DNA(comprising a coding sequence for MAD7 nuclease under the control of thepL inducible promoter, a chloramphenicol resistance gene, and the λ Redrecombineering system) was added to 50 μL “FtsY shuttle vector” E. colicells. The transformed cells were plated on LB plates with 25 μg/mLchloramphenicol (chlor) and 100 μg/mL carbenicillin and incubatedovernight to accumulate clonal isolates. The next day, a colony waspicked, grown overnight in LB+25 μg/mL chlor+100 μg/mL carb, andglycerol stocks were prepared from the saturated overnight culture byadding 500 μL 50% glycerol to 1000 μL culture. The stocks of “FtsYshuttle vector” E. coli cells comprising the engine vector were frozenat −80° C.

Transformation with Editing Library: The assembled editing vector andelectrocompetent “FtsY shuttle vector” E. coli cells (that carry boththe target polynucleotide on a shuttle vector and the engine vector)were transferred into a transformation module for electroporation. Thetransformation module comprised an ADP-EPC cuvette. See, e.g., U.S. PatNo. 62/551,069. The cells and nucleic acids were combined and allowed tomix for 1 minute, and electroporation was performed for 30 seconds. Theparameters for the poring pulse were: voltage, 2400 V; length, 5 ms;interval, 50 ms; number of pulses, 1; polarity, +. The parameters forthe transfer pulses were: Voltage, 150 V; length, 50 ms; interval, 50ms; number of pulses, 20; polarity, +/−. Following electroporation, thecells were transferred to a recovery module (another growth module) andallowed to recover in SOC medium containing kanamycin. Carbenicillin andchloramphenicol were added to the medium after 1 hour, and the cellswere allowed to recover for another 2 hours. After recovery, the cellswere held at 4° C. until recovered by the user. An aliquot of cells wasplated on LB agar supplemented with chloramphenicol, carbenicillin, andkanamycin.

Hundreds of colonies were picked on a high-throughput roboticcolony-picking apparatus, grown in LB+ampicillin (100 μg/mL) in 96-wellformat, and edited shuttle vector DNA was prepared by 96-well formatminiprep. Isolated shuttle vector DNA was sequenced to identify edits.

For identified mutations, the integrating segment of the edited shuttlevector was liberated from the pDG1662 backbone by restriction enzymedigestion of approximately 1 μg of shuttle vector DNA with AatII andBstXI. Restriction enzymes were heat-inactivated prior to B. subtilistransformation.

A single colony of B. subtilis was inoculated into 4.5 mL of Medium A(81 mL sterile water; 10 mL 10× Medium A base; 10× Bacillus salts; seeYasbin et al. Transformation and Transfection in Lysogenic Strains ofBacillus subtilis: Evidence for Selective Induction of Prophage inCompetent Cells. Journal of Bacteriology. 121:1 296-304. 1975.) andadjusted with additional inoculants to reach an OD₆₅₀ of 0.1-0.2.Culture was incubated at 37° C. with vigorous shaking from 60-90 minutesand OD₆₅₀ was recorded every 20 minutes. Culture was grown for 90minutes after the cessation of logarithmic growth. 0.05 mL of thisculture was transferred into 0.45 mL of pre-warmed Medium B, one tubefor each transformation, incubated at 37° C. with vigorous shaking for90 minutes, after which cultures were highly competent. 1 μg of digestededited shuttle vector DNA was added to each transformation tube, one foreach edit, and incubated at 37° C. with shaking for 30 minutes. Aliquotsof transformed B. subtilis cells were plated onto agar plates containingchloramphenicol (5 μg/mL).

Individual B. subtilis transformants carrying the integrated editedshuttle vector were picked, propagated in culture, and impact of editsof the FtsY gene were evaluated in a high-throughput functional assay.

Example #2

Cloning human genetic locus into TAR shuttle vector in yeast:Saccharomyces cerevisiae strain VL6-48 (MAT alpha, his3-Δ200, trp1-Δ1,ura3-Δ1, lys2, ade2-101, met14) that has HIS3, TRP1, and URA3 deletedwas used as a host for TAR cloning of human gene LMNA (ATCC NumberMYA-3666™).

The TAR cloning vector pVC-604 (ATCC® MBA-212™) contains a yeastselectable marker (HIS3) and a yeast centromeric sequence (CEN6). Beforeuse, the TAR vector was “activated” by insertion of the targetingsequences (hooks) specific for LMNA (5′ and 3′ ends of the gene) intothe polylinker. Such shuttle vector should be constructed before TARcloning experiments. Hooks were unique sequences; no repeated sequenceswere present in the hooks. The uniqueness of hooks was checked byBLASTing against the human genome. The minimal size of the targetinghooks was 60 bp. Before TAR cloning, the vector was linearized bycutting between the targeting hooks with a restriction enzyme.Concentration of the linearized TAR vector was 0.5-1 μg/μl. Vector DNAwas isolated by DNA Maxi kit (Qiagen).

A yeast-bacteria-mammalian cell shuttle vector for retrofitting circularYAC/TAR isolates into a BAC form (“retrofitting vector”) contained a 3′HPRT-loxP-eGFP cassette, allowing gene loading into a unique loxP sitewithin the HAC-based shuttle vector. An F′ factor origin of replicationallows YAC propagation as a single copy BAC molecule.

The Cre recombinase expression vector was derived from thepCpGfree-vitroHLacZ plasmid (InvivoGen). It contained a hygromycinresistance gene that allows selection of the plasmid in both bacteriaand vertebrate cells. The optimized Cre gene is under the EF1 promoterand flanked by Matrix Attachment Region (MAR) elements. The MAR elementstogether with the absence of CpG sites in promoter or vector sequencesgreatly improve the transcription efficiency of the Cre gene. The iCreplasmid also contains a conditional R6K bacterial origin, which requiresa pir+expressing E. coli strain to propagate.

Highly competent yeast spheroplasts were prepared by pelleting anovernight culture of Saccharomyces cerevisiae strain VL6-48,resuspending in 20 mL of 1 M Sorbitol, pelleting again, resuspending in20 mL of SPE solution (1 M Sorbitol, 10 mM Nae EDTA, 0.01 M Naphosphate, pH 7.5), adding 20 μL zymolyase solution (10 mg/ml ofzymolyase 20T in 20% glycerol), 40 μL of ME (14.3 Mbeta-mercaptoethanol), mixing, and incubating at 30° C. for 20 minutes.Spheroplasts were centrifuged for 10 min at 570× g at 5° C. Supernatantwas decanted, cells gently resuspended in 50 ml of 1.0 M Sorbitol, andthe spheroplasts were pelleted again by centrifugation for 10 min at300-600× g at 5° C. The 50 ml of 1 M Sorbitol wash was repeated one moretime, and the final pellet was gently resuspended in 2.0 ml of STCsolution (1 M Sorbitol, 10 mM CaCl₂, 10 mM Tris-HCl, pH 7.5).

200 μL of competent spheroplast suspension was mixed gently with 1-2 μgof human genomic DNA and 1.0 μg of linearized shuttle vector pVC-604 andincubated for 10 minutes at room temperature. 800 μL of PEG 8000solution was added and gently mixed by inverting and incubated at roomtemperature for 10 minutes. Spheroplasts were pelleted by centrifugationfor 5 min at 300-500× g at 5° C. Supernatant was removed andspheroplasts were gently resuspended in 800 μl of SOS solution (1 MSorbitol, 6.5 mM CaCl₂), 0.25% yeast extract, 0.5% Bacto Peptone) bypipetting. Spheroplasts were incubated for 40 minutes at 30° C. withoutshaking.

Spheroplasts were transferred to a 15-ml Falcon conical tube containing7.0 ml of melted TOP agar-His (1 M Sorbitol, 2% D-glucose, 0.17% YeastNitrogen Base, 0.5% (NH₄)₂SO₄, 3% agar containing the followingsupplements: 0.006% adenine sulfate, 0.006% uracil, 0.005%L-arginine.HCl, 0.008% L-aspartic acid, 0.01% L-GLUTAMIC acid, 0.005%L-isoleucine, 0.01% L-leucine, 0.012% L-lysine.HCl, 0.002% L-methionine,0.005% L-phenylalanine, 0.0375% L-serine, 0.01% L-threonine, 0.005%L-tryptophan, 0.005% L-tyrosine, 0.015% L-valine equilibrated at 50° C.)by pipetting, gently mixed, and agar mix was quickly poured onto aSORB-His plate (1 M Sorbitol, 2% D-glucose, 0.17% Yeast Nitrogen Base0.5% (NH₄)₂SO₄, 2% agar supplemented as described above) with selectivemedium containing 1 M Sorbitol. Plates were incubated at 30° C. for 5-7days until transformants became visible.

Positive clones were identified and confirmed by isolating shuttlevector DNA by standard techniques, amplifying across the insertjunctions with PCR primers designed complementary to the shuttle vectorsequence flanking the target polynucleotide insert, and sequencing thejunctions.

Editing Target Polynucleotide in Yeast: A positive clone carrying theYAC shuttle vector described above was rendered electrocompetent andco-transformed with a library of editing cassettes directed towardsaturation mutagenesis of the target polynucleotide and an editingvector backbone by electroporation. In vivo recombination aftertransformation results in a complete editing vector carrying thecomponents necessary for nucleic acid-guided nuclease editing describedherein. Growth on selective medium allows for propagation only of cellsthat carry a complete editing vector and have successfully received anedit of the target polynucleotide.

Retrofitting Edited YAC Shuttle Vector Into BAC: 5 ml of SD-Hissynthetic liquid medium without histidine was inoculated with oneindividual colony containing the edited TAR/YAC shuttle vector and grownovernight at 30° C. with vigorous shaking. The culture was transferredinto 50-mL liquid YPD and grown for an additional 4-5 hours at 30° C.with vigorous shaking. The culture was pelleted by centrifugation for 5minutes at 1,000×g at 5° C. in a 50-mL Falcon tube, and supernatant wasdiscarded. Pellet was resuspended in 10 mL of sterile water byvortexing, transferred into an Eppendorf tube, and pelleted again. Cellswere resuspended in 10 mL of LiAc solution (100 mM lithium acetate, 10mM Tris-HCl, 0.1 mM EDTA, pH 7.5), incubated at 30° C. for 1 hour withslow shaking, and collected by centrifugation. Supernatant was discardedand cells were resuspended in 100 μL LiAc solution by pipetting. 1 μg ofBamHI linearized “retrofitting vector” DNA and 5 μL of carrier salmonDNA (10 mg/ml sonicated salmon sperm DNA (Stratagene) denaturated byboiling for 10 min every time before the experiment) were added to cellsand mixed well. 0.45 mL of fresh PEG 4000 solution was added to cells,mixed by vortexing, and incubated for 1 hour at 30° C. Cells wereheat-shocked in a 42° C. heating block for 15 minutes. Cells were rinsedwith sterile water, collected by centrifugation at high speed for 1minutes, and supernatant was discarded. Cells were rinsed one more timewith sterile water, supernatant was discarded, cells were resuspended insterile water and the suspension was plated on SD-Ura plates. Plateswere incubated for 2-3 days at 30° C. until Ura colonies were observed.Ura transformants now contained the retrofitted YAC/BAC shuttle vectorcarrying the edited target polynucleotide.

Transferring Retrofitted Shuttle Vector from Yeast to E. coli: Two tothree His+Ura+transformants carrying the edited shuttle vector wereinoculated separately in 5 ml of YPD medium each in 50-ml Falcon conicaltubes and grown overnight at 30° C. with a vigorous shaking. Cells werepelleted and resuspended in 100 μl EDTA mix (0.05 M EDTA, 0.01 MTris-HCl, pH 7.5) and transferred to 1.5 mL Eppendorf tubes. 100 μl ofzymolyase solution was added, mixed by vortexing, and incubated for 30minutes at 37° C. Cell suspension and equal volumes low melting pointagarose (LMP: 1% of agarose gel prepared in 0.125 M EDTA, pH 7.5) wereequilibrated in a heat block at 42° C. Agarose plugs of the cellsuspension were made by mixing cell suspension and LMP 1:1 and usingpipette tips as molds. Solidified agarose plugs were suspended in LETsolution (0.5 M EDTA, 0.01 M Tris-HCl, pH 7.5) in Eppendorf tubes andincubated for 1 hour at 37° C. LET solution was removed, plugs werecovered in NDS cell lysis buffer (0.39 M EDTA, 0.01 M Tris-HCl, pH 7.5,1% N-lauroyl sarcosine, 2 mg/ml proteinase K), and incubated for 1 hourat 55° C. NDS buffer was removed and plugs were washed three times withEDTA mix. To electroporate the edited YAC/BAC shuttle vector into E.coli, the plugs were melted at 68° C. for 15 min, cooled to 42° C. for10 min, treated with 1.5 units of β-agarase for 1 h at 42° C., andchilled on ice for 10 minutes. The plugs were diluted twofold withsterile water, and 1 μl of the mixture was used to electroporate 20 μlof electrocompetent E. coli cells. After electroporation cells wererecovered in SOC medium for 1 hour at 37° C. Cells were plated onLB-Chlor plates and incubated at 37° C. overnight.

Insertion of Edited Target Nucleotide from Shuttle Vector into HAC: Toinsert the edited target nucleotide into the HAC, the retrofittedYAC/BAC shuttle vector was co-transfected with a Cre-recombinaseexpression vector into a hprt-minus hamster CHO cells carrying the HACand HPRT-plus colonies were selected onHypoxanthine-Aminopterin-Thymidine (HAT) medium. YAC/BAC shuttle vectorDNA was prepared by Large-Construct Kit (Qiagen) and Cre-recombinaseexpression plasmid by Spin Miniprep Kit (Qiagen), respectively. One daybefore transfection, CHO cells were plated in a six-well plate of 2 mlof growth medium (F12+10% FBS+1% PenStrep+8 μg/ml blasticidin). 20-30 μgof prepared YAC/BAC shuttle vector DNA and 1 μg of Cre-recombinaseexpression vector DNA were diluted in 100 μL Opti-MEM medium withoutserum. 10 μL of Lipofectamine 2000 was diluted in 90 μL of Opti-MEM.Diluted DNAs and diluted Lipofectaimine were combined for a total volumeof 200 μL and incubated at room temperature for 20-30 minutes. At thesame time CHO cells were washed in six-well plate one time by PBS,rinsed with 2 ml of Opti-MEM medium, aspirated and washed with 2 mlOpti-MEM medium again (three washes in total), and incubated at 37° C.for 20-30 minutes. After incubation cells the Opti-MEM wash wasaspirated from cells, 800 μL of Opti-MEM was added to the DNA mixture(total volume now 1 mL), the diluted DNA mixture was applied to cells,and incubated for 12 hours at 37° C. Cells were washed with growth mediaone to two times and incubated for 12-16 h at 37° C. in 2 ml of growthmedia. Cells were seeded in 10-cm plates and F12-HAT media was added(F12+10% FBS+1% PenStrep+8 μg/ml blasticidin+1×HAT). Colonies were grownin HAT selection for 2-3 weeks and picked using cloning cylinders at0.25% Trypsin. Colonies were transferred to a six-well plate and laterto 10-cm plates for further colony expansion. For each positive colony,cells were collected from confluent 10-cm plates to make frozen stocksand prepare HAC DNA using standard genomic DNA purification protocols.Positive clones were confirmed by PCR and sequencing across the insertjunction to verify that the edited target polynucleotide wassuccessfully transferred to the HAC.

HAC DNA carrying the edited target polynucleotide was transfected intohuman cells lines for functional studies and further characterization.

Example #3

Producing a Drosophila melanogaster Genomic BAC Library

Drosophila melanogaster genomic DNA was prepared from adult flies bystandard techniques. DNA was partially digested with EcoRI and EcoRImethylase, size fractionated, and cloned into the pBACe3.6 vector(BACPAC Resources Center, Children's Hospital Oakland ResearchInstitute, Oakland, Calif.) by ligation with T4 DNA ligase andtransformation into E. coli. Digested genomic DNA was ligated withlinearized vector at an approximately 1:10 molar ratio of insert:vector.Average insert size was approximately 160 kb. Individual colonies werepicked and arrayed in 96-well plates and grown in 25 μg/mLchloramphenicol (chlor) to yield a genomic library of 17,620 recombinantclones representing approximately 25× coverage of the Drosophila genome.BAC DNA was prepared by high-throughput 96-well format miniprep andpair-end sequenced to identify inserts. Collection of inserts wasreduced to represent approximately 1× overlapping coverage of theDrosophila melanogaster genome.

Editing Cassette Preparation: 5 nM of oligonucleotides directed towardsaturation mutagenesis of the Drosophila melanogaster genome synthesizedon a chip were amplified using Q5 polymerase in 50 μL volumes. The PCRconditions were 95° C. for 1 minute; 8 rounds of 95° C. for 30seconds/60° C. for 30 seconds/72° C. for 2.5 minutes; with a final holdat 72° C. for 5 minutes. Following amplification, the PCR products weresubjected to SPRI cleanup, where 30 μL SPRI mix was added to the 50 μLPCR reactions and incubated for 2 minutes. The tubes were subjected to amagnetic field for 2 minutes, the liquid was removed, and the beads werewashed 2× with 80% ethanol, allowing 1 minute between washes. After thefinal wash, the beads were allowed to dry for 2 minutes, 50 μL 0.5× TEpH 8.0 was added to the tubes, and the beads were vortexed to mix. Theslurry was incubated at room temperature for 2 minutes, then subjectedto the magnetic field for 2 minutes. The eluate was removed, and the DNAquantified.

Following quantification, a second amplification procedure was carriedout using a dilution of the eluate from the SPRI cleanup. PCR wasperformed under the following conditions: 95° C. for 1 minute; 18 roundsof 95° C. for 30 seconds/72° C. for 2.5 minutes; with a final hold at72° C. for 5 minutes. Amplicons were checked on a 2% agarose gel andpools with the cleanest output(s) were identified. Amplificationproducts appearing to have heterodimers or chimeras were not used.

Backbone Preparation: A 10-fold serial dilution series of purifiedbackbone was performed, and each of the diluted backbone series wasamplified under the following conditions: 95° C. for 1 minute; then 30rounds of 95° C. for 30 seconds/60° C. for 1.5 minutes/72° C. for 2.5minutes; with a final hold at 72° C. for 5 minutes. After amplification,the amplified backbone was subjected to SPRI cleanup as described abovein relation to the cassettes. The backbone was eluted into 100 μL ddH₂Oand quantified before Gibson Assembly®.

Isothermal Assembly: 150 ng backbone DNA was combined with 100 ngcassette DNA. An equal volume of 2× Master Mix was added, and thereaction was incubated for 45 minutes at 50° C. After assembly, theassembled backbone and cassettes were subjected to SPRI cleanup, asdescribed above.

Transformation with Engine Vector: 1 μL of the engine vector DNA(comprising a coding sequence for MAD7 nuclease under the control of thepL inducible promoter, an ampicillin resistance gene, and the λ Redrecombineering system) was added to 50 μL E. coli cells in each well ofan array of 384-well plates representing the genomic library describedabove by liquid handling automation. The transformed cells were platedon LB plates with 25 μg/mL chloramphenicol (chlor) and 100 μg/mLcarbenicillin and incubated overnight to accumulate clonal isolates. Thenext day, a colony was picked, grown overnight in LB+25 μg/mL chlor+100μg/mL carb, and glycerol stocks were prepared from the saturatedovernight culture by adding 500 μL 50% glycerol to 1000 μL culture. Thecollection of stocks of E. coli cells comprising the Drosophila genomicBAC library and engine vector were frozen at −80° C.

Transformation with Editing Library: The assembled editing vector andelectrocompetent collection of E. coli cells carrying the Drosophilagenomic BAC library (that carry both the target polynucleotide on ashuttle vector and the engine vector) were transferred into atransformation module for electroporation. The transformation modulecomprised an ADP-EPC cuvette. See, e.g., U.S. Pat No. 62/551,069. Thecells and nucleic acids were combined and allowed to mix for 1 minute,and electroporation was performed for 30 seconds. The parameters for theporing pulse were: voltage, 2400 V; length, 5 ms; interval, 50 ms;number of pulses, 1; polarity, +. The parameters for the transfer pulseswere: Voltage, 150 V; length, 50 ms; interval, 50 ms; number of pulses,20; polarity, +/−. Following electroporation, the cells were transferredto a recovery module (another growth module) and allowed to recover inSOC medium containing kanamycin. Carbenicillin and chloramphenicol wereadded to the medium after 1 hour, and the cells were allowed to recoverfor another 2 hours. After recovery, the cells were held at 4° C. untilrecovered by the user. An aliquot of cells was plated on LB agarsupplemented with chloramphenicol, carbenicillin, and kanamycin.

Preparation of Edited Shuttle Vector DNA: Edited BAC DNA was prepared byhigh-throughput 96-well format miniprep. For each clone, edited targetpolynucleotide was liberated from the BAC backbone and subcloned in aninsect expression vector for downstream functional studies in Drosophilacells. Alternatively, BAC DNA may be isolated from the editing E. colipopulation in bulk and transfected into Drosophila cells in bulk.

Example #4

Targeted Insertion of Heterologous DNA in the Saccharomyces cerevisiaeGenome

Targeted insertion of a single-copy of a heterologous DNA sequence (geneor pathway) into the genome of yeast was achieved by taking advantage ofthe endogenous homology-directed repair machinery of Saccharomycescerevisiae to precisely integrate said DNA into the desired location onthe target chromosome. One system for precise integration ofheterologous DNA employs addition to the 5′ and 3′ ends of theheterologous DNA sequence homology regions of identical sequence to thetargeted location of the yeast genome. Additionally, a recyclablecounter-selectable marker gene such as URA3 was also included in thetargeting DNA sequence. By flanking the counter-selectable marker with ashort direct repeat sequence, the marker can be removed from the finalintegrated DNA construct, leaving behind in the genome only the desiredheterologous DNA sequence for further editing with the automatedmulti-module cell processing system. Transforming DNA was constructedfully intact by gene synthesis though alternatively the DNA may beassembled from two or more fragments using strand overlap extension(SOE) PCR or other assembly methods. Assembled DNA was then transformedinto S. cerevisiae and integrated into the yeast chromosome byhomologous recombination and candidate strains were obtained by platingon selective growth media, uracil dropout media in this example. Oncethe correct genomic integration of the heterologous DNA construct wasconfirmed, the URA3 marker was removed by plating colonies onto growthmedium containing 5-fluoroorotic acid (5-FOA) and screening for clonesin which the URA3 marker cassette has looped out between the flankingdirect repeats. The markerless strain is now ready for genome editing onthe heterologous DNA. The outcome of one such editing experiment wasevaluated and the data is shown in FIGS. 9 and 10. FIG. 9 is a tableshowing the edit category and the number of wells that were in each editcategory. Note that there were 44 wells comprising clonal completeintended edits, and 6 wells with subclonal complete intended edits. FIG.10 is a bar graph showing the edit categories.

Example #5 Fully-Automated Singleplex RGN-directed Editing Run

Singleplex automated genomic editing using MAD7 nuclease wassuccessfully performed with an automated multi-module instrument of thedisclosure. See U.S. Pat. No. 9,982,279; and U.S. Ser. No. 16/024,831filed 30 Jun. 2018; Ser. No. 16/024,816 filed 30 Jun. 2018; Ser. No.16/147,353 filed 28 Sep. 2018; Ser. No. 16/147,865 filed 30 Sep. 2018;and Ser. No. 16/147,871 filed 30 Jun. 2018.

An ampR plasmid backbone and a lacZ_F172* editing cassette wereassembled via Gibson Assembly® into an “editing vector” in an isothermalnucleic acid assembly module included in the automated instrument.lacZ_F172 functionally knocks out the lacZ gene. “lacZ_F172*” indicatesthat the edit happens at the 172nd residue in the lacZ amino acidsequence. Following assembly, the product was de-salted in theisothermal nucleic acid assembly module using AMPure beads, washed with80% ethanol, and eluted in buffer. The assembled editing vector andrecombineering-ready, electrocompetent E. Coli cells were transferredinto a transformation module for electroporation. The cells and nucleicacids were combined and allowed to mix for 1 minute, and electroporationwas performed for 30 seconds. The parameters for the poring pulse were:voltage, 2400 V; length, 5 ms; interval, 50 ms; number of pulses, 1;polarity, +. The parameters for the transfer pulses were: Voltage, 150V; length, 50 ms; interval, 50 ms; number of pulses, 20; polarity, +/−.Following electroporation, the cells were transferred to a recoverymodule (another growth module), and allowed to recover in SOC mediumcontaining chloramphenicol. Carbenicillin was added to the medium after1 hour, and the cells were allowed to recover for another 2 hours. Afterrecovery, the cells were held at 4° C. until recovered by the user.

After the automated process and recovery, an aliquot of cells was platedon MacConkey agar base supplemented with lactose (as the sugarsubstrate), chloramphenicol and carbenicillin and grown until coloniesappeared. White colonies represented functionally edited cells, purplecolonies represented un-edited cells. All liquid transfers wereperformed by the automated liquid handling device of the automatedmulti-module cell processing instrument.

The result of the automated processing was that approximately 1.0E⁰³total cells were transformed (comparable to conventional benchtopresults), and the editing efficiency was 83.5%. The lacZ_172 edit in thewhite colonies was confirmed by sequencing of the edited region of thegenome of the cells. Further, steps of the automated cell processingwere observed remotely by webcam and text messages were sent to updatethe status of the automated processing procedure.

Example #6 Fully-Automated Recursive Editing Run

Recursive editing was successfully achieved using the automatedmulti-module cell processing system. An ampR plasmid backbone and alacZ_V10* editing cassette were assembled via Gibson Assembly® into an“editing vector” in an isothermal nucleic acid assembly module includedin the automated system. Similar to the lacZ_F172 edit, the lacZ_V10edit functionally knocks out the lacZ gene. “lacZ_V10” indicates thatthe edit happens at amino acid position 10 in the lacZ amino acidsequence. Following assembly, the product was de-salted in theisothermal nucleic acid assembly module using AMPure beads, washed with80% ethanol, and eluted in buffer. The first assembled editing vectorand the recombineering-ready electrocompetent E. Coli cells weretransferred into a transformation module for electroporation. The cellsand nucleic acids were combined and allowed to mix for 1 minute, andelectroporation was performed for 30 seconds. The parameters for theporing pulse were: voltage, 2400 V; length, 5 ms; interval, 50 ms;number of pulses, 1; polarity, +. The parameters for the transfer pulseswere: Voltage, 150 V; length, 50 ms; interval, 50 ms; number of pulses,20; polarity, +/−. Following electroporation, the cells were transferredto a recovery module (another growth module) allowed to recover in SOCmedium containing chloramphenicol. Carbenicillin was added to the mediumafter 1 hour, and the cells were grown for another 2 hours. The cellswere then transferred to a centrifuge module and a media exchange wasthen performed. Cells were resuspended in TB containing chloramphenicoland carbenicillin where the cells were grown to OD600 of 2.7, thenconcentrated and rendered electrocompetent.

During cell growth, a second editing vector was prepared in anisothermal nucleic acid assembly module. The second editing vectorcomprised a kanamycin resistance gene, and the editing cassettecomprised a galK_Y145* edit. If successful, the galK_Y145* edit conferson the cells the ability to uptake and metabolize galactose. The editgenerated by the galK_Y154* cassette introduces a stop codon at the154th amino acid reside, changing the tyrosine amino acid to a stopcodon. This edit makes the galK gene product nonfunctional and inhibitsthe cells from being able to metabolize galactose. Following assembly,the second editing vector product was de-salted in the isothermalnucleic acid assembly module using AMPure beads, washed with 80%ethanol, and eluted in buffer. The assembled second editing vector andthe electrocompetent E. Coli cells (that were transformed with andselected for the first editing vector) were transferred into atransformation module for electroporation, using the same parameters asdetailed above. Following electroporation, the cells were transferred toa recovery module (another growth module), allowed to recover in SOCmedium containing carbenicillin. After recovery, the cells were held at4° C. until retrieved, after which an aliquot of cells were plated on LBagar supplemented with chloramphenicol, and kanamycin. To quantify bothlacZ and galK edits, replica patch plates were generated on two mediatypes: 1) MacConkey agar base supplemented with lactose (as the sugarsubstrate), chloramphenicol, and kanamycin, and 2) MacConkey agar basesupplemented with galactose (as the sugar substrate), chloramphenicol,and kanamycin. All liquid transfers were performed by the automatedliquid handling device of the automated multi-module cell processingsystem.

In this recursive editing experiment, 41% of the colonies screened hadboth the lacZ and galK edits, the results of which were comparable tothe double editing efficiencies obtained using a “benchtop” or manualapproach.

While this invention is satisfied by embodiments in many differentforms, as described in detail in connection with preferred embodimentsof the invention, it is understood that the present disclosure is to beconsidered as exemplary of the principles of the invention and is notintended to limit the invention to the specific embodiments illustratedand described herein. Numerous variations may be made by persons skilledin the art without departure from the spirit of the invention. The scopeof the invention will be measured by the appended claims and theirequivalents. The abstract and the title are not to be construed aslimiting the scope of the present invention, as their purpose is toenable the appropriate authorities, as well as the general public, toquickly determine the general nature of the invention. In the claimsthat follow, unless the term “means” is used, none of the features orelements recited therein should be construed as means-plus-functionlimitations pursuant to 35 U.S.C. § 112, ¶6.

We claim:
 1. A method of nucleic acid-guided nuclease editing of fivehundred or more exogenous target polynucleotides from source cellswithin heterologous editing cells in a single multiplexed automatedoperation, comprising: inserting five hundred or more exogenous targetpolynucleotides from the source cells into shuttle vector backbones toform a library of shuttle vectors; transforming heterologous editingcells with the library of shuttle vectors to produce first transformedheterologous editing cells; transferring the first transformedheterologous editing cells into a growth module; growing the firsttransformed heterologous editing cells in a growth module; transferringthe first transformed heterologous editing cells from the growth moduleto a transformation module; providing one or more editing vectors in afirst receptacle wherein the editing vectors comprise a coding sequencefor a nuclease, a guide nucleic acid and a DNA donor sequence, andwherein the one or more editing vectors target one or more of theexogenous target polynucleotides; transferring the one or more editingvectors from the first receptacle to the transformation module;introducing the one or more editing vectors into the first transformedheterologous editing cells in the transformation module to producesecond transformed heterologous editing cells; transferring the secondtransformed heterologous editing cells from the transformation module toan editing module; allowing editing to take place in the editing moduleunder conditions that allow the editing vectors to edit the exogenoustarget polynucleotides in the second transformed heterologous editingcells thereby forming edited cells; isolating the edited cells; andisolating the edited shuttle vectors; wherein the first receptacle,growth module, transformation module and editing module are all part ofa stand-alone automated multi-module cell processing instrument, andwherein a liquid handling system moves the editing vectors from thefirst receptacle to the transformation module, and moves the firsttransformed heterologous editing cells from the growth module to thetransformation module and from the transformation module to the editingmodule all without human intervention.
 2. The method of claim 1, whereinone or more of the five hundred or more exogenous target polynucleotidesare selected from a full-length gene; an open reading frame codingsequence; or a genomic locus of size 1000-10,000 nucleotides, 50-500nucleotides, 10-100 nucleotides, or 10,000-100,000 nucleotides.
 3. Themethod of claim 2, wherein one or more of the five hundred or moreexogenous target polynucleotides are single open reading frame codingsequences.
 4. The method of claim 2, wherein one or more of the fivehundred or more exogenous target polynucleotides are genomic loci ofsize 1000-10,000 nucleotides.
 5. The method of claim 1, wherein thesource cells are selected from bacterial cells, fungal cells, plantcells, mammalian cells and human cells.
 6. The method of claim 5,wherein the source cells are bacterial cells.
 7. The method of claim 5,wherein the source cells are fungal cells.
 8. The method of claim 5,wherein the source cells are plant cells.
 9. The method of claim 5,wherein the source cells are mammalian cells.
 10. The method of claim 1,shuttle vector backbones are selected from bacterial plasmids, yeastcentromeric plasmids, bacterial artificial chromosomes, yeast artificialchromosomes, BAC/YAC hybrids and mammalian artificial chromosomes. 11.The method of claim 10, wherein the shuttle vector backbone is abacterial plasmid.
 12. The method of claim 10, wherein the shuttlevector backbone is a yeast centromeric plasmid.
 13. The method of claim10, wherein the shuttle vector backbone is a bacterial artificialchromosome.
 14. The method of claim 10, wherein the shuttle vectorbackbone is a yeast artificial chromosome.
 15. The method of claim 10,wherein the shuttle vector is a mammalian artificial chromosome.
 16. Themethod of claim 1, wherein the editing cells are selected from bacterialcells, fungal cells, plant cells, mammalian cells and human cells or acombination thereof.
 17. The method of claim 1, wherein the editingvectors confer edits comprising saturation mutagenesis edits, knockoutedits, loss of function edits, gain of function edits, coding sequenceedits and noncoding sequence edits.
 18. The method of claim 17, whereinthe editing vectors confer edits comprising saturation mutagenesisedits.
 19. The method of claim 17, wherein the editing vectors conferknockout edits.
 20. The method of claim 17, wherein the editing vectorsconfer loss of function edits.
 21. The method of claim 17, wherein theediting vectors confer coding sequence edits.
 22. The method of claim17, wherein the editing vectors confer noncoding sequence edits.
 23. Themethod of claim 1, wherein the stand-alone automated multi-module cellprocessing instrument further comprises a cell concentration module, andfurther comprising the step of after growing the first transformedheterologous editing cells in the growth module and before transferringthe first transformed heterologous editing cells from the growth moduleto the transformation module, the first transformed heterologous editingcells are concentrated in the cell concentration module.
 24. The methodof claim 23, wherein the cell concentration module performs tangentialflow filtration.
 25. The method of claim 1, wherein the step ofintroducing the library of shuttle vectors into the heterologous editingcells is performed using a flow-through electroporation (FTEP) device.26. The method of claim 1, wherein the growth module comprises arotating growth module.
 27. The method of claim 1, wherein thestand-alone automated multi-module cell processing instrument furthercomprises a recovery module.
 28. The method of claim 1, wherein thestand-alone automated multi-module cell processing instrument iscontrolled by a processor.
 29. The method of claim 1, wherein one ormore of the five hundred or more different target polynucleotides areselected from single open reading frame coding sequences, a genesequence from 5′UTR to 3′UTR, and a genetic locus comprising at least100 kb.